Epstein Barr virus induced genes

ABSTRACT

The present invention relates, in general, to Epstein Barr Virus induced (EBI) genes. In particular, the present invention relates to DNA segments coding for EBI 1, EBI 2, or EBI 3 polypeptide; EBI 1, EBI 2, or EBI 3 polypeptide; recombinant DNA molecules; cells containing the recombinant DNA molecules; antisense EBI 1, EBI 2, or EBI 3 constructs; antibodies having binding affinity to an EBI 2, EBI 2, or EBI 3 polypeptide; hybridomas containing the antibodies; nucleic acid probes for the detection of the presence of Epstein Barr Virus; a method for detecting Epstein Barr Virus in a sample; and kits containing nucleic acid probes or antibodies.

RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.09/929,583, filed on Aug. 14, 2001, now pending; which is a continuationof application Ser. No. 09/536,954, filed on Mar. 28, 2000, and nowissued as U.S. Pat. No. 6,500,926; which is a divisional of applicationSer. No. 08/352,678, filed on Nov. 30, 1994 and now issued as U.S. Pat.No. 6,043,351; which was a continuation of application Ser. No.07/980,518, filed on Nov. 25, 1992, and now abandoned.

STATEMENT OF GOVERNMENT RIGHTS IN THE INVENTION

[0002] Part of the work performed during development of this inventionutilized U.S. Government funds. The U.S. Government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates, in general, to Epstein Barr Virusinduced (EBI) genes. In particular, the present invention relates to DNAsegments coding for EBI 1, EBI 2, or EBI 3 polypeptides; EBI 1, EBI 2,or EBI 3 polypeptides; recombinant DNA molecules; cells containing therecombinant DNA molecules; antisense EBI 1, EBI 2, or EBI 3 constructs;antibodies having binding affinity to an EBI 1, EBI 2, or EBI 3polypeptide; hybridomas containing the antibodies; nucleic acid probesfor the detection of the presence of Epstein Barr Virus; a method ofdetecting Epstein Barr Virus in a sample; and kits containing nucleicacid probes or antibodies.

[0005] 2. Background Information

[0006] Epstein-Barr Virus (EBV) is the cause of infectiousmononucleosis, a benign proliferation of infected B lymphocytes (Henle,G., et al., Proc. Natl. Acad. Sci. USA, 59(1):94-101 (1968)) and canalso cause acute and rapidly progressive B lymphoproliferative diseasein severely immune compromised patients or in experimental infection oftamarins (Miller, G., Fields Virol., 2nd ed., 1921-58 (1990)). Infectionof human B lymphocytes, in vitro, results in expression of six virusencoded nuclear proteins (EBNAs) and two virus encoded membrane proteins(LMPs) (Kieff and Liebowitz, Fields Virol., 2nd ed., 1889-1920 (1990)),and in substantially altered cell growth (Nilsson and Klein, Adv. CancerRes. 37(319):319-80 (1982)). EBV infected B lymphocytes recapitulatefeatures of antigen stimulation in enlarging, increasing RNA synthesis,expressing activation antigens and adhesion molecules, secreting Ig andproliferating (Boyd, A. W., et al., J. Immunol. 134(3): 1516-23 (1985);Gordon, J., et al., Immunology, 58(4):591-5 (1986); Guy and Gordon,Intl. J. Cancer 43(4):703-8 (1989); Nilsson and Klein, Adv. Cancer Res.37(319):319-80 (1982); Thorley-Lawson, D. A., et al., J. Immunol.134(5):3007-12 (1985)). Unlike antigen stimulated B lymphocytes, EBVinfected B lymphocytes continue to proliferate in vitro as immortalizedlymphoblastoid cell lines (LCLs) (Nilsson, K., et al., Intl. J. Cancer8(3):443-50 (1971)).

[0007] EBV effects on lymphocytes have been studied by comparing theproperties of EBV-negative [EBV(−)] Burkitt lymphoma (BL) cell lines andEBV-positive [EBV(+)] derivatives, infected by EBV, in vitro (Calendar,A., et al., Proc. Natl. Acad. Sci. USA 84(22):8060-4 (1987);Ehlin-Henriksson, B., et al., Intl. J. Cancer 39(2):211-8 (1987);Nilsson and Klein, Adv. Cancer Res. 37(319):319-80 (1982); Rowe, M., etal., Intl. J. Cancer 37(3):367-73 (1986)). EBV(−) BL cells resembleproliferating centroblasts of germinal centers, characteristicallyexpressing CD10, CD20, CD77 (BLA), class II antigen, and thecarbohydrate recognized by peanut agglutinin (Calendar, A., et al.,Proc. Natl. Acad. Sci. USA 84(22):8060-4); Ehlin-Henriksson, B., et al.,Intl. J. Cancer 39(2):211-8 (1987); Favrot, M. C., et al., Intl. J.Cancer 38(6):901-6 (1986); Gregory, C. D., et al., Intl. J. Cancer42(2):213-20 (1988); Gregory, C. D., et al., J. Gen. Virol.71:1481-1495(1990); Gregory, C. D., et al., J. Immunol. 139(1):313-8(1987); Rowe, M., et al., Intl. J. Cancer 37(3):367-73 (1986); Rowe, M.,et al., Intl. J. Cancer 35(4):435-41 (1985)). Both EBV(−) BL cells andcentroblasts lack surfage IgD and antigens associated with early phasesof mitogen stimulation in vitro, including CD23, CD39 and CD30. Ingeneral, EBV(+) BL cells closely resembly EBV infected primary Blymphocytes in not expressing CD10 or CD77 and in expressing earlyactivation and differentiation markers, vimentin, Bac-1, Bcl-2, surfaceIgD and CD44 (Calendar, A., et al., Proc. Natl. Acad. Sci. USA84(22):8060-4 (1987); Ehlin-Henriksson, B., et al., Intl. J. Cancer39(2):211-8 (1987); Favrot, M. C., et al., Intl. J. Cancer 38(6):901-6(1986); Gregory, C. D. e, al., J. Gen. Virol. 71:1481-1495 (1990);Henderson, S., et al., Cell 65(7):1107-15 (1991); Rowe, M., et al.,Intl. J. Cancer 37(3):367-73 (1986); Rowe, M., et al., EMBO J,6(9):2743-51 (1987); Spira, G., et al., J. Immunol. 126(1):122-6 (1981);Suzuki, T., et al., J. Immunol. 137(4):1208-13 (1986)). Experiments withsingle gene transfer into EBV(−) B lymphoma cells, or with specificallymutated EBV recombinants reveal that EBNA 2, LMP 1 and EBNA 3C areessential for lymphocyte growth transformation and alter cellular orviral gene expression. Expression of EBNA 2 alone in EBV(−) BL celllines results in enhanced transcription of CD23, CD21 (Cordier, M., etal., J. Virol. 64(3):1002-13 (1990); Wang, F., et al., J. Virol.64(5):2309-18 (1990); Wang, F., et al., Proc. Natl. Acad. Sci. USA84(10):3452-6 (1987)), and c-fgr (Knutson, J. C., J. Virol. 64(6):2530-6(1990)). EBNA 2 also transactivates the LMP promoters (Fahraeus, R., etal., Proc. Natl. Acad. Sci. USA 87(19):7390-4 (1990); Wang, F., et al.,J. Virol. 64(7):3407-16 (1990)). Analysis of a series of EBNA 2 mutantsindicates that the ability of EBNA 2 to transactivate gene expression istightly linked to essential role in cell growth transformation (Cohen,J. I., et al., J. Virol. 65(5):2545-54 (1991)). LMP 1 is also criticalto EBV's effects on cell growth. LMP 1 transforms immortalized rodentfibroblasts (Baichwal and Sugden, Oncogene 2(5):461-7 (1988); Wang, D.,et al., Cell 43:831-40 (1985)) and includes vimentin, Bcl-2 and many ofthe activation markers and adhesion molecules that EBV induces in BLcells (Birkenbach, M., et al., J. Virol. 63(9):4079-84 (1989);Henderson, S., et al., Cell 65(7):1107-15 (1991); Wang, D., et al., J.Virol. 62(11):4173-84 (1988)). In EBV(−) BL cells, EBNA 3c induceshigher level expression of CD21 (Wang, F., et al., J. Virol.64(5):2309-18 (1990)).

[0008] Since altered B lymphocyte gene expression is a central theme inEBV induced changes in B lymphocyte growth, a more complete descriptionof the repertoire of EBV induced genes would be advantageous prior tothe investigation of specific genes for their role as mediators of EBVeffects on cell growth. Also, because of the similar effects of EBV andantigen, EBV induced genes are likely to include mediators of antigeninduced B lymphocyte growth or differentiation. Previously, recognitionof such genes has been largely based on increased expression oflymphocyte surface markers (Calendar, A., et al., Proc. Natl. Acad. Sci.USA 84(22):8060-4 (1987)), defined by monoclonal antibodies derivedagainst EBV or antigen activated B lymphocytes. Few of these surfacemarkers are likely candidates for important effectors of EBV or antigenincuded alterations in lymphocyte growth. The experiments described hereuse subtractive hybridization to identify cDNA clones of RNAs which aremore abundant in an in vitro infected EBV(+) BL cell than in thenon-infected EBV(−) control BL cell.

SUMMARY OF THE INVENTION

[0009] It is a general object of this invention to provide EBI 1, EBI 2,and EBI 3 DNA segments. It is a specific object of this invention toprovide a DNA segment coding for a polypeptide having an amino acidsequence corresponding to an EBI 1, EBI 2, or EBI 3 polypeptide.

[0010] It is another object of the invention to provide a substantiallypure polypeptide having an amino acid sequence corresponding to an EBI1, EBI 2, or EBI 3 polypeptide.

[0011] It is a further object of the invention to provide a nucleic acidprobe for the detection of the presence of Epstein Barr Virus in asample.

[0012] It is another object of the invention to provide a method ofdetecting Epstein Barr Virus in a sample.

[0013] It is a further emobdiment of the invention to provide a kit foridentifying or amplifying a gene encoding an EBI 1, EBI 2, or EBI 3polypeptide.

[0014] It is another object of the invention to provide a DNA moleculecomprising, 5′ to 3′, a promoter effective to initiate transcription ina cell and an EBI 1, EBI 2, or EBI 3 DNA segment.

[0015] It is a further object of the invention to provide a recombinantDNA molecule comprising a vector and an EBI 1, EBI 2, or EBI 3 DNAsegment.

[0016] It is a further object of the invention to provide a DNA moleculecomprising a transcriptional region functional in a cell, a sequencecomplimentary to an RNA sequence encoding an amino acid sequencecorresponding to an EBI 1, EBI 2, or EBI 3 polypeptide, and atranscriptional termination region functional in said cell.

[0017] It is another object of the invention to provide cells containingthe above-described DNA molecules.

[0018] It is a further object of the invention to provide an antibodyhaving binding affinity to an EBI 1, EBI 2, or EBI 3 polypeptide, or abinding fragment thereof.

[0019] It is another object of the invention to provide a hybridomawhich produces the above-described antibody, or binding fragmentthereof.

[0020] It is a further object of the invention to provide a method ofdetecting an EBI 1, EBI 2, or EBI 3 polypeptide in a sample.

[0021] It is another object of the invention to provide a diagnostic kitcomprising EBI 1, EBI 2, or EBI 3 antibodies.

[0022] Further objects and advantages of the present invention will beclear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIGS. 1A and 1B. EBV induced gene (EBI) 1 and 2 RNA: Nucleotideand deduced amino acid sequences. (FIG. 1A) EBI 1 (nucleotide sequenceis SEQ ID NO:1, amino acid sequence is SEQ ID NO:2) has two potentialtranslational initiation codons. In frame stop codons are indicatedasterisks (*). A hydrophobic amino terminal segment (single underline)is predicted to be a signal peptide for membrane translocation. Sevenother highly hydrophobic segments are predicted to form membranespanning domains and are delineated by double underlines. Potentialasparagine linked glycosylation sites (CHO######) are present in theextracellular amino terminal segment and third extracellular loop. Thesequence motif S-(I/V)-D-R-(Y/F)-X-X-X-X (SEQ ID NO:7) where Xrepresents consecutive hydrophobic residues), is highly conserved amonga large number of G-protein coupled receptors and is indicated at theend of the third transmembrane domain (::::). (FIG. 1B) EBI 2(nucleotide sequence is SEQ ID NO:3, amino acid sequence is SEQ ID NO:4)has 2 possible initiator methionine codons. Predicted transmembranedomains are indicated (double underlines). No signal peptide sequencewas identified. The amino terminal extracellular segment contains apotential N-linked glycosylation site (CHO######).

[0024]FIG. 2. RNA blot hybridization analysis of EBV induced cellulargene expression. Polyadenylylated (4 to 12 μg per lane) was sizefractionated on formaldehyde agarose gels, transferred to charged nylonmembranes, and hybridized with the probes indicated at the bottom ofeach autoradiograph panel. RNA samples used are indicated at the top ofeach lane (LCL:EBV immortalized primary B lymphoblastoid cell line,1134; BL:EBV negative Burkitt lymphoma cell line, BL41; EBL:EBV infectedBurkitt lymphoma cell line, BL41/B95-8, derived by in vitro infection ofBL41 line). Dashes indicate positions of ribosomal RNA bands (18 s, 28s). The band detected at 1.5 kb in the LCL lane by the P68 probe is dueto residual signal from a prior hybridization.

[0025]FIG. 3. Expression of EBI 1 and EBI 2 receptor genes in humanlymphoid tissues and cell lines. ³²P-labelled probes indicated at theleft of each panel were hybridized to blots containing RNA from the celllines indicated at the top of each lane. BL41 and BL30 are EBV-negativeBL cell lines; BL41/P3HR1 is infected with a non-transforming EBVstrain, p3HR1; BL41/B95-8 is infected with a transforming EBV strain;IB4 is a cell line derived by infecting primary B lymphocytes with EBVof the B95-8 strain; LCL-W91 is a recently established cell linetransformed with EBV strain W91; TONSIL is unfractionated cells fromsurgically excised human tonsil; PBMC is unfractionated peripheral bloodmononuclear cells; PBMC PWM is PBMC stimulated 72 h with pokeweedmitogen (2.5 μg/ml); PBT PHA is T cells purified from PBMC by sheeperythrocyte rosetting, stimulated 72 h with phytohemagglutinin (1μg/ml); B MARR is post-mortem bone marrow; SPLEEN is unfractionatedcells from surgically excised spleen; HL60 is a promyelocytic leukemiacell line; U937 is a monocytic leukemia cell line; K62 is a chronicmyelogenous leukemia cell line; JURKAT is a T cell leukemia cell line;HSB-2 is a T cell acute lymphoblastic leukemia cell line; RHEK-1 is anadenovirus/SV40 transformed human keratinocyte; TK143 is a osteosarcomacell line. Each panel is a composite prepared from autoradiographs oftwo separate blots for each probe.

[0026]FIG. 4. EBI 1 and EBI 2 gene expression in human tissues. EBI 1,EBI 2 and immunoglobulin mu chain (IgU) probes were hybridized to RNAsamples from the following human tissues; heart (HE), brain (BR),placenta (PL), lung (LU), liver (LI), kidney (KI), skeletal muscle (SM)and pancreas (PA). Numbers at the left indicate positions and sizes (inkb) of RNA markers. Specific RNA bands are indicated by arrows to theright of each panel. The EBI 1 probe detects faint 2.4 kb bands in lungand pancreas PNA. The EBI 2 probe detects an abundant 1.9 kb RNA inlung, and a faint 1.9 kb band in pancreas. The 2.7 kb IgU RNA isdetected in lung, liver and pancreas preparations. The 1.5 kb band inplacental RNA hybridized with IgU probe is residual signal from aprevious hybridization.

[0027] FIGS. 5A-C. Complete nucleotide and deduced amino acid sequencesof EBI 3 cDNA (nucleotide sequence is SEQ ID NO:5, amino acid sequenceis SEQ ID NO:6). The 1164 nucleotide EBI 3 cDNA (SEQ ID NO:5) contains a690 nucleotide open reading frame encoding a 26 kD polypeptide (SEQ IDNO:6). A hydrophobic amino terminal segment (bold underline) comprises asignal peptide for membrane translocation. No other hydrophobic segmentsthat could potentially form a transmembrane domain are evident. Twopotential asparagine-linked glycosylation sites are indicated (CHO###).The nucleotide sequence of the 3′ untranslated region bears significanthomology with the human Alu repeat element (light underline).

[0028]FIG. 6. RNA blot hybridization analysis of EBI 3 gene expression.Polyadenylated RNA (4 to 12 μg/lane) was size fractionated onformaldehyde agarose gel, transferred to an activated nylon membrane andhybridized with a ³²P-labeled EBI 3 cDNA, actin and glyceraldehydedehydrogenase (GAPDH) probes. RNA samples used in each lane areindicated at the top. (LCL is the EBV-immortalized primary Blymphoblastoid cell line, IB4; BL is the EBV-negative Burkitt lymphomacell line, BL41; EBL is the EBV-infected Burkitt lymphoma cell line,BL41/B95-8, derived by in vitro infection of BL41 line). An abundant 1.5kb RNA is recognized by the EBI 3 probe in both EBV-infected cell lineRNA samples (LCL, EBL), but is undetectable in the EBV-negative cellsample (BL). Control hybridization with actin and GAPDH probes indicatethat the BL lane contains as much or more RNA than the EBV-infected celllanes. Dashes indicate positions of ribosomal RNA bands (18 s, 28 s).

[0029]FIGS. 7A and 7B. Expression of EBI 3 gene RNA in human tissues andcell lines.

[0030] (FIG. 7A) EBI 3 or actin probes were hybridized to blotscontaining RNA from the cell lines or lymphoid tissues indicated at thetop of each lane. (BL41 and BL30 are EBV(−) Burkitt lymphoma cell lines;BL41/P3HR1 is infected with the non-transforming P3HR1 strain of EBV;BL41/B95-8 is infected with the transforming, B95-8 strain of EBV; IB4is a lymphoblastoid cell line generated by transformation of primary Blymphocytes with B95-8 virus; LCL-W91 EBV strain; TONSIL representsunfractionated cells from surgically excised human tonsil; PBMC isunfractionated peripheral blood mononuclear cells; PBMC-PWM is PBMCstimulated 72 h with pokeweed mitogen (2.5 μg/ml); PBT-PHA is T cellspurified from PBMC by sheep erythrocyte rosetting, stimulated 72 h withphytohemagglutinin (1.0 μg/mL); B MARR is post-mortem costal bonemarrow; SPLEEN is unfractionated cells from surgically excised normalhuman spleen; HL60 is a promyelocytic leukemia cell line; U937 is ahistiocytic lymphoma cell line with monocyte features; K562 is a chronicmyelogenous leukemia cell line; Jurkat is a T cell leukemia; TK143 is anosteosarcoma line. Each autoradiographic panel was generated from twoseparate blots.

[0031] (FIG. 7B) EBI 3 was hybridized to a commercially prepared blot(Multiple Tissue Northern, Clontech, CA) containing polyadenylated RNA(2 μg/lane) from each of the following human tissues: heart (HE), brain(BR), placenta (PL), lung (LU), liver (LI), kidney (KI), skeletal muscle(SM) and pancreas (PA). The EBI 3 probe specifically detects an abundant1.5 kb RNA in the placental RNA preparation (position indicated byarrow). A faint band of similar size is also observed in liver RNA.Numbers at the left indicate positions and sizes (in kb) of RNA markers.

DEFINITIONS

[0032] In the description that follows, a number of terms used inrecombinant DNA (rDNA) technology are extensively utilized. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

[0033] DNA segment. A DNA segment, as is generally understood and usedherein, refers to a molecule comprising a linear stretch of nucleotideswherein the nucleotides are present in a sequence that may encode,through the genetic code, a molecule comprising a linear sequence ofamino acid residues that is referred to as a protein, a protein fragmentor a polypeptide.

[0034] Gene. A DNA sequence related to a single polypeptide chain orprotein, and as used herein includes the 5′ and 3′ untranslated ends.The polypeptide can be encoded by a full-length sequence or any portionof the coding sequence, so long as the functional activity of theprotein is retained.

[0035] A “complimentary DNA” or “cDNA” gene includes recombinant genessynthesized by reverse transcription of messenger RNA (“mRNA”).

[0036] Structural gene. A DNA sequence that is transcribed into mRNAthat is then translated into a sequence of amino acids characteristic ofa specific polypeptide.

[0037] Restriction Endonuclease. A restriction endonuclease (alsorestriction enzyme) is an enzyme that has the capacity to recognize aspecific base sequence (usually 4, 5, or 6 base pairs in length) in aDNA molecule, and to cleave the DNA molecule at every place where thissequence appears. For example, EcoRI recognizes the base sequenceGAATTC/CTTAAG.

[0038] Restriction Fragments. The DNA molecules produced by digestionwith a restriction endonuclease are referred to as restrictionfragments. Any given genome may be digested by a particular restrictionendonuclease into a discrete set of restriction fragments.

[0039] Agarose Gel Electrophoresis. To detect a polymorphism in thelength of restriction fragments, an analytical method for fractionatingdouble-stranded DNA molecules on the basis of size is required. The mostcommonly used technique (though not the only one) for achieving such afractionation is agarose gel electrophoresis. The principle of thismethod is that DNA molecules migrate through the gel as though it were asieve that retards the movement of the largest molecules to the greatestextent and the movement of the smallest molecules to the least extent.Note that the smaller the DNA fragment, the greater the mobility underelectrophoresis in the agarose gel.

[0040] The DNA fragments fractionated by agarose gel electrophoresis canbe visualized 10 directly by a staining procedure if the number offragments included in the pattern is small. The DNA fragments of genomescan be visualized successfully. However, most genomes, including thehuman genome, contain far too many DNA sequences to produce a simplepattern of restriction fragments. For example, the human genome isdigested into approximately 1,000,000 different DNA fragments by EcoRI.In order to visualize a small subset of these fragments, a methodologyreferred to as the Southern hybridization procedure can be applied.

[0041] Southern Transfer Procedure. The purpose of the Southern transferprocedure (also referred to as blotting) is to physically transfer DNAfractionated by agarose gel electrophoresis into a nitrocellulose filterpaper or another appropriate surface or method, while retaining therelative positions of DNA fragments resulting from the fractionationprocedure. The methodology used to accomplish the transfer from agarosegel to nitrocellulose involves drawing the DNA from the gel into thenitrocellulose paper by capillary action.

[0042] Nucleic Acid Hybridization. Nucleic acid hybridization depends onthe principle that two single-stranded nucleic acid molecules that havecomplementary base sequences will reform the thermodynamically favoreddouble-stranded structure if they are mixed under the proper conditions.The double-stranded structure will be formed between two complementarysingle-stranded nucleic acids even if one is immobilized on anitrocellulose filter. In the Southern hybridization procedure, thelatter situation occurs. As noted previously, the DNA of the individualto be tested is digested with a restriction endonuclease, fractionatedby agarose gel electrophoresis, converted to the single-stranded form,and transferred to nitrocellulose paper, making it available forreannealing to the hybridization probe.

[0043] Hybridization Probe. The visualize a particular DNA sequence inthe Southern hybridization procedure, a labeled DNA molecule orhybridization probe is reacted to the fractionated DNA bound to thenitrocellulose filter. The areas in the filter that carry DNA sequencescomplementary to the labeled DNA probe become labeled themselves as aconsequence of the reannealing reaction. The areas of the filter thatexhibit such labeling are visualized. The hybridization probe isgenerally produced by molecular cloning of a specific DNA sequence.

[0044] Oligonucleotide or Oligomer. A molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three. Itsexact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. An oligonucleotide maybe derived synthetically or by cloning.

[0045] Sequence Amplification. A method for generating large amounts ofa target sequence. In general, one or more amplification primers areannealed to a nucleic acid sequence. Using appropriate enzymes,sequences found adjacent to, or in between the primers are amplified.

[0046] Amplification Primer. An oligonucleotide which is capable ofannealing adjacent to a target sequence and serving as an initiationpoint for DNA synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is initiated.

[0047] Vector. A plasmid or phage DNA or other DNA sequence into whichDNA may be inserted to be cloned. The vector may replicate autonomouslyin a host cell, and may be further characterized by one or a smallnumber of endonuclease recognition sites as which such DNA sequences maybe cut in a determinable fashion and into which DNA may be inserted. Thevector may further contain a marker suitable for use in theidentification of cells transformed with the vector. Markers, forexample, are tetracycline resistance or ampicillin resistance. The words“cloning vehicle” are sometimes used for “vector.”

[0048] Expression. Expression is the process by which a structural geneproduces a polypeptide. It involves transcription of the gene into mRNA,and the translation of such mRNA into polypeptide(s).

[0049] Expression vector. A vector or vehicle similar to a cloningvector but which is capable of expressing a gene which has been clonedinto it, after transformation into a host. The cloned gene is usuallyplaced under the control of (i.e., operably linked to) certain controlsequences such as promoter sequences.

[0050] Expression control sequences will vary depending on whether thevector is designed to express the operably linked gene in a prokaryoticor eukaryotic host and may additionally contain transcriptional elementssuch as enhancer elements, termination sequences, tissue-specificityelements, and/or translational initiation and termination sites.

[0051] Functional Derivative. A “functional derivative” of a sequence,either protein or nucleic acid, is a molecule that possesses abiological activity (either functional or structural) that issubstantially similar to a biological activity of the protein or nucleicacid sequence. A functional derivative of a protein may or may notcontain post-translational modifications such as covalently linkedcarbohydrate, depending on the necessity of such modifications for theperformance of a specific function. The term “functional derivative” isintended to include the “fragments,” “segments,” “variants,” “analogs,”or “chemical derivatives” of a molecule.

[0052] As used herein, a molecule is said to be a “chemical derivative”of another molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties may improve themolecule's solubility, absorption, biological half life, and the like.The moieties may alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, andthe like. Moieties capable of mediating such effects are disclosed inRemington's Pharmaceutical Sciences (1980). Procedures for coupling suchmoieties to a molecule are well known in the art.

[0053] Fragment. A “fragment” of a molecule such as a protein or nucleicacid is meant to refer to any portion of the amino acid or nucleotidegenetic sequence.

[0054] Variant. A “variant” of a protein or nucleic acid is meant torefer to a molecule substantially similar in structure and biologicalactivity to either a protein or nucleic acid, or to a fragment thereof.Thus, provided that two molecules possess a common activity and maysubstitute for each other, they are considered variants as that term isused herein even if the composition or secondary, tertiary, orquaternary structure of one of the molecules is not identical to thatfound in the other, or if the amino acid or nucleotide sequence is notidentical.

[0055] Analog. An “analog” of a protein or genetic sequence is meant torefer to a protein or genetic sequence substantially similar in functionto a protein or genetic sequence described herein.

[0056] Allele. An “allele” is an alternative form of a gene occupying agiven locus on the chromosome.

[0057] Mutation. A “mutation” is any detectable change in the geneticmaterial which may be transmitted to daughter cells and possibly even tosucceeding generations giving rise to mutant cells or mutantindividuals. If the descendants of a mutant cell give rise only tosomatic cells in multicellular organisms, a mutant spot or area of cellsarises. Mutations in the germ line of sexually reproducing organisms maybe transmitted by the gametes to the next generation resulting in anindividual with the new mutant condition in both its somatic and germcells. A mutation may be any (or a combination of) detectable, unnaturalchange affecting the chemical or physical constitution, mutability,replication, phenotypic function, or recombination or one or moredeoxyribonucleotides; nucleotides may be added, deleted, substitutedfor, inverted, or transposed to new positions with and withoutinversion. Mutations may occur spontaneously and can be inducedexperimentally by application of mutagens. A mutant variation of a DNAsegment results from a mutation. A mutant polypeptide may result from amutant DNA segment.

[0058] Species. A “species” is a group of actually or potentiallyinterbreeding natural populations. A species variation within a DNAsegment or protein is a change in the nucleic acid or amino acidsequence that occurs among species and may be determined by DNAsequencing of the segment in question.

[0059] Substantially Pure. A “substantially pure” protein or nucleicacid is a protein or nucleic acid preparation that is generally lackingin other cellular components.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention relates to novel DNA sequences, EBI 1, EBI2, and EBI 3, which have been identified as Epstein Barr Virus inducedgenes.

[0061] A. DNA Segments for Coding for EBI 1 EBI 2, and EBI 3Polypeptides, and Fragments Thereof.

[0062] In one embodiment, the present invention relates to a DNA segmentcoding for a polypeptide having an amino acid sequence corresponding toa polypeptide selected from the group consisting of EBI 1, EBI 2, andEBI 3 polypeptides, or at least 7 contiguous amino acids thereof(preferably, at least 10, 15, 20, or 30 contiguous amino acids thereof).In one preferred embodiment, the DNA segment comprises the sequences setforth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; allelic, mutant orspecies variation thereof, or at least 20 contiguous nucleotides thereof(preferably at least 25, 30, 40, or 50 contiguous nucleotides thereof).In another preferred embodiment, the DNA segment encodes an amino acidsequence selected from the group consisting of sequences set forth inSEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, or mutant or speciesvariation thereof, or at least 7 contiguous amino acids thereof(preferably, at least 10, 15, 20, or 30 contiguous amino acids thereof).

[0063] Also included within the scope of this invention are thefunctional equivalents of the herein-described DNA or nucleotidesequences. The degeneracy of the genetic code permits substitution ofcertain codons by other codons which specify the same amino acid andhence would give rise to the same protein. The DNA or nucleotidesequence can vary substantially since, with the exception of methionineand tryptophan, the known amino acids can be coded for by more than onecodon. Thus, portions or all of the EBI 1, EBI 2, or EBI 3 gene could besynthesized to give a DNA sequence significantly different from thatshown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. The encoded aminoacid sequence thereof would, however, be preserved.

[0064] In addition, the DNA or nucleotide sequence may comprise anucleotide sequence which results from the addition, deletion orsubstitution of at least one nucleotide to the 5′-end and/or the 3′-endof the DNA formula shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 ora derivative thereof. Any nucleotide or polynucleotide may be used inthis regard, provided that its addition, deletion or substitution doesnot alter the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 4, or SEQID NO: 6 which is encoded by the nucleotide sequence. For example, thepresent invention is intended to include any nucleotide sequenceresulting from the addition of ATG as an initiation codon at the 5′-endof the inventive nucleotide sequence or its derivative, or from theaddition of TTA, TAG or TGA as a termination codon at the 3′-end of theinventive nucleotide sequence or its derivative. Moreover, the DNAfragment of the present invention may, as necessary, have restrictionendonuclease recognition sites added to its 5′-end and/or 3′-end.

[0065] Such functional alterations of a given DNA or nucleotide sequenceafford an opportunity to promote secretion and/or processing ofheterologous proteins encoded by foreign DNA sequences fused thereto.All variations of the nucleotide sequence of the EBI 1, EBI 2, and EBI 3genes and fragments thereof permitted by the genetic code are,therefore, included in this invention.

[0066] Further, it is possible to delete codons or to substitute one ormore codons by codons other than degenerate codons to produce astructurally modified polypeptide, but one which has substantially thesame utility or activity of the polypeptide produced by the unmodifiedDNA molecule. As recognized in the art, the two polypeptides arefunctionally equivalent, as are the two DNA molecules which give rise totheir production, even though the differences between the DNA moleculesare not related to degeneracy of the genetic code.

[0067] A. 1. Isolation of DNA.

[0068] In one aspect of the present invention, DNA segments coding forpolypeptides having amino acid sequences corresponding to EBI 1, EBI 2,and EBI 3 are provided. In particular, the DNA segment may be isolatedfrom a biological sample containing RNA or DNA.

[0069] The DNA segment may be isolated from a biological samplecontaining RNA using the techniques of cDNA cloning and subtractivehybridization as previously described (Birkenbach et al., J. of Virology63:9:4079-4084). The DNA segment may also be isolated from a cDNAlibrary using a homologous probe.

[0070] The DNA segment may be isolated from a biological samplecontaining genomic DNA or from a genomic library using techniques wellknown in the art. Suitable biological samples include, but are notlimited to, blood, semen and tissue. The method of obtaining thebiological sample will vary depending upon the nature of the sample.

[0071] One skilled in the art will realize that the human genome may besubject to slight allelic variations between individuals. Therefore, theisolated DNA segment is also intended to include allelic variations, solong as the sequence is a functional derivative of the EBI 1, EBI 2, orEBI 3 gene.

[0072] One skilled in the art will realize that organisms other thanhumans may also contain EBI 1, EBI 2, or EBI 3 genes (for example,eukaryotes; more specifically, mammals, birds, fish, and plants; morespecifically, gorillas, rhesus monkeys, and chimpanzees). The inventionis intended to include, but not be limited to, EBI 1, EBI 2, and EBI 3DNA segments isolated from the above-described organisms.

[0073] A.2. Synthesis of DNA.

[0074] In the alternative, the DNA segment of the present invention maybe chemically synthesized. For example, a DNA fragment with thenucleotide sequence which codes for the expression product of an EBI 1,EBI 2, or EBI 3 gene may be designed and, if necessary, divided intoappropriate smaller fragments. Then an oligomer which corresponds to theDNA fragment, or to each of the divided fragments, may be synthesized.Such synthetic oligonucleotides may be prepared, for example, by thetriester method of Matteucci et al., J. Am. Chem. Soc. 103:3185-3191(1981) or by using an automated DNA synthesizer.

[0075] An oligonucleotide may be derived synthetically or by cloning. Ifnecessary, the 5′-ends of the oligomers may be phosphorylated using T4polynucleotide kinase. Kinasing of single strands prior to annealing orfor labeling may be achieved using an excess of the enzyme. If kinasingis for the labeling of probe, the ATP may contain high specificityactivity radioisotopes. Then, the DNA oligomer may be subjected toannealing and ligation with T4 ligase or the like.

[0076] B. A Substantially Pure EBI 1, EBI 2, and EBI 3 Polypeptide.

[0077] In another embodiment, the present invention relates to asubstantially pure polypeptide having an amino acid sequencecorresponding to a polypeptide selected from the group consisting of EBI1, EBI 2, and EBI 3 polypeptides, or at least 7 contiguous amino acidsthereof (preferably, at least 10, 15, 20, or 30 contiguous amino acidsthereof). In a preferred embodiment, the polypeptide has an amino acidsequence selected from the group consisting of sequences set forth inSEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, or mutant or speciesvariation thereof, or at least 7 contiguous amino acids thereof(preferably, at least 10, 15, 20, or 30 contiguous amino acids thereof).

[0078] A variety of methodologies known in the art can be utilized toobtain the peptide of the present invention. In one embodiment, thepeptide is purified from tissues or cells which naturally produce thepeptide. The samples of the present invention include cells, proteinextracts or membrane extracts of cells, or biological fluids. The samplewill vary based on the assay format, the detection method and the natureof the tissues, cells or extracts used as the sample.

[0079] Any eukaryotic organism can be used as a source for the peptideof the invention, as long as the source organism naturally contains sucha peptide. As used herein, “source organism” refers to the originalorganism from which the amino acid sequence of the subunit is derived,regardless of the organism the subunit is expressed in and ultimatelyisolated from.

[0080] One skilled in the art can readily follow known methods forisolating proteins in order to obtain the peptide free of naturalcontaminants. These include, but are not limited to:immunochromatography, size-exclusion chromatography, HPLC, ion-exchangechromatography, and immuno-affinity chromatography.

[0081] C. A nucleic Acid Probe for the Detection of Epstein Barr Virus.

[0082] In another embodiment, the presnet invention relates to a nucleicacid probe for the detection of the presence of Epstein Barr Virus in asample comprising the above-described DNA segments or at least 20contiguous nucleotides thereof (preferably at least 25, 30, 40, or 50thereof). In another preferred embodiment, the DNA segment has a nucleicacid sequence selected from the group consisting of sequences set forthin SEQ ID NO:1, SEQ ID NO: 3, and SEQ ID NO:5, or at least 20 contiguousnucleotides thereof (preferably at least 25, 30, 40, or 50 thereof). Inanother preferred embodiment, the nucleic acid probe encodes an aminoacid sequence selected from the group consisting of sequences set forthin SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, or at least 7 contiguousamino acids thereof.

[0083] The nucleic acid probe may be used to probe an appropriatechromosomal or cDNA library by usual hybridization methods to obtainanother DNA segment of the present invention. A chromosomal DNA or cDNAlibrary may be prepared from appropriate cells according to recognizedmethods in the art (cf. Molecular Cloning: A Laboratory Manual, secondedition, edited by Sambrook, Fritsch & Maniatis, Cold Spring HarborLaboratory, 1989).

[0084] In the alternative, chemical synthesis is carried out in order toobtain nucleic acid probes having nucleotide sequences which correspondto N-terminal and C-terminal portions of the amino acid sequence of thepolypeptide of interest. Thus, the synthesized nucleic acid probes maybe used as primers in a polymerase chain reaction (PCR) carried out inaccordance with recognized PCR techniques, essentially according to PCRProtocols, A Guide to Methods and Applications, edited by Michael etal., Academic Press, 1990, utilizing the appropriate chromosomal or cDNAlibrary to obtain the fragment of the present invention.

[0085] One skilled in the art can readily design such probes based onthe sequence disclosed herein using methods of computer alignment andsequence analysis known in the art (cf. Molecular Cloning: A LaboratoryManual, second edition, edited by Sambrook, Fritsch & Maniatis, ColdSpring Harbor Laboratory, 1989).

[0086] The hybridization probes of the present invention can be labeledby standard labeling techniques such as with a radiolabel, enzyme label,fluorescent label, biotin-avidin label, chemiluminescence, and the like.After hybridization, the probes may be visualized using known methods.

[0087] The nucleic acid probes of the present invention include RNA, aswell as DNA probes, such probes being generated using techniques knownin the art.

[0088] In one embodiment of the above-described method, a nucleic acidprobe is immobilized on a solid support. Examples of such solid supportsinclude, but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, and acrylic resins, such aspolyacrylamide and latex beads. Techniques for coupling nucleic acidprobes to such solid supports are well known in the art.

[0089] The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The sample used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

[0090] D. A Method of Detecting the Presence of Epstein Barr Virus in aSample.

[0091] In another embodiment, the present invention relates to a methodof detecting the presence of Epstein Barr Virus in a sample comprising(a) contacting said sample with the above-described nucleic acid probe,under conditions such that hybridization occurs, and (b) detecting thepresence of said probe bound to said DNA segment. One skilled in the artwould select the nucleic acid probe according to techniques known in theart as described above. Samples to be tested include but should not belimited to RNA samples of human tissue. The presence of EBI 1, EBI 2, orEBI 3 may represent that cells had been infected with the Epstein BarrVirus. Increases in the amount of EBI 1, EBI 2, or EBI 3 RNA in a samplemay also indicate the presence of or infection with the Epstein BarrVirus.

[0092] E. A kit for Detecting the Presence of Epstein Barr Virus in aSample.

[0093] In another embodiment, the present invention relates to a kit fordetecting the presence of Epstein Barr Virus in a sample comprising atleast one container means having disposed therein the above-describednucleic acid probe. In a preferred embodiment, the kit further comprisesother containers comprising one or more of the following: wash reagentsand reagents capable of detecting the presence of bound nucleic acidprobe. Examples of detection reagents include, but are not limited to,radiolabelled probles, enzymatic labeled probes (horse radishperoxidase, alkaline phosphatase), and affinity labeled probes (biotin,avidin, or steptavidin).

[0094] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers or strips of plastic orpaper. Such containers allow the efficient transfer of reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated and the agents or solutions of each containercan be added in a quantitative fashion from one compartment to another.Such containers will include a container which will accept the testsample, a container which contains the probe or primers used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, and the like), and containers whichcontain the reagents used to detect the hybridized probe, boundantibody, amplified product, or the like.

[0095] One skilled in the art will readily recognize that the nucleicacid probes described in the present invention can readily beincorporated into one of the established kit formats which are wellknown in the art.

[0096] F. DNA Constructs Comprising the EBI 1, EBI 2, or EBI 3 DNASegments and Cells containing these constructs.

[0097] In another embodiment, the present invention relates to arecombinant DNA molecule comprising, 5′ to 3′, a promoter effective toinitiate transcription in a host cell and the above-described DNAsegments.

[0098] In another embodiment, the present invention relates to arecombinant DNA molecule comprising a vector and an above-described DNAsegment.

[0099] In another embodiment, the present invention relates to a DNAmolecule comprising a transcriptional region functional in a cell, asequence complementary to an RNA sequence encoding an amino acidsequence corresponding to the above-described polypeptide, and atranscriptional termination region functional in said cell.

[0100] Preferably, the above-described molecules are isolated and/orpurified DNA molecules.

[0101] In another embodiment, the present invention relates to a cellthat contains an above-described DNA molecule.

[0102] In another embodiment, the peptide is purified from cells whichhave been altered to express the peptide.

[0103] As used herein, a cell is said to be “altered to express adesired peptide” when the cell, through genetic manipulation, is made toproduce a protein which it normally does not produce or which the cellnormally produces at low levels. One skilled in the art can readilyadapt procedures for introducing and expressing either genomic, cDNA, orsynthetic sequences into either eukaryotic or prokaryotic cells.

[0104] A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but shall in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

[0105] If desired, the non-coding region 3′ to the sequence encoding anEBI 1, EBI 2, or EBI 3 gene may be obtained by the above-describedmethods. This region may be retained for its transcriptional terminationregulatory sequences, such as termination and polyadenylation. Thus, beretaining the 3′-region naturally contiguous to the DNA sequenceencoding an EBI 1, EBI 2, or EBI 3 gene, the transcriptional terminationsignals may be provided. Where the transciptional termination signalsare not satisfactorily functional in the expression host cell, then a 3′region functional in the host cell may be substituted.

[0106] Two DNA sequences (such as a promoter region sequence and an EBI1, EBI 2, or EBI 3 sequence) are said to be operably linked if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region sequence to direct the transcription ofan EBI 1, EBI 2, EBI 3 gene sequence, or (3) interfere with the abilityof an EBI 1, EBI 2, or EBI 3 gene sequence to be transcribed by thepromoter region sequence. Thus, a promoter region would be operablylinked to a DNA sequence if the promoter were capable of effectingtranscription of that DNA sequence.

[0107] Thus, to express an EBI 1, EBI 2, or EBI 3 gene, transcriptionaland translational signals recognized by an appropriate host arenecessary.

[0108] The present invention encompasses the expression of the EBI 1,EBI 2, or EBI 3 gene (or a functional derivative thereof) in eitherprokaryotic or eukaryotic cells. Prokaryotic hosts are, generally, themost efficient and convenient for the production of recombinant proteinsand, therefore, are preferred for the expression of the EBI 1, EBI 2, orEBI 3 gene.

[0109] Prokaryotes most frequently are represented by various strains ofE. coli. However, other microbial strains may also be used, includingother bacterial strains.

[0110] In prokaryotic systems, plasmid vectors that contain replicationsites and control sequences derived from a species compatible with thehost may be used. Examples of suitable plasmid vectors may includepBR322, pUC18, pUC19 and the like; suitable phage or bacteriophagevectors may include λgt10, λgt11, and the like; and suitable virusvectors may include pMAM-neo, pKRC and the like. Preferably, theselected vector of the present invention has the capacity to replicatein the selected host cell.

[0111] Recognized prokaryotic hosts include bacteria such as E. coli,Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like.However, under such conditions, the peptide will not be glycosylated.The prokaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

[0112] To express EBI 1, 2, or 3 (or a functional derivative thereof) ina prokaryotic cell, it is necessary to operably link the EBI 1, EBI 2,or EBI 3 sequence to a functional prokaryotic promoter. Such promotersmay be either constitutive or, more preferably, regulatable (i.e.,inducible or derepressible). Examples of constitutive promoters includethe int promoter of bacteriophage λ, the bla of the β-lactamase genesequence of pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (PL and PR), the trp, recA, lacz, lacI, and galpromoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol.162:176-182 (1985)) and the ç-specific promoters of B. subtilis (Gilmanet al., Gene sequence 32:11-20 (1984)), the promoters of thebacteriophages of Bacillus (Gryczan, In: The Molecular Biology of theBacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters(Ward et al., Mol. Gen. Genet. 203:468-478 (1986)).

[0113] Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol.1:277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986); andGottesman (Ann. Rev. Genet. 18:415-442 (1984)).

[0114] Proper expression in a prokaryotic cell also requires thepresence of a ribosome binding site upstream of the genesequence-encoding sequence. Such ribosome binding sites are disclosed,for example, by Gold et al., (Ann. Rev. Microbiol. 35:365-404 (1981)).

[0115] The selection of control sequences, expression vectors,transformation methods, and the like, are dependent on the type of hostcell used to express the gene. As used herein, “cell”, “cell line”, and“cell culture” may be used interchangeably and all such designationsinclude progeny. Thus, the words “transformants” or “transformed cells”include the primary subject cell and cultures derived therefrom, withoutregard to the number of transfers. It is also understood that allprogeny may not be precisely identical in DNA content, due to deliberateor inadvertent mutations. However, as defined, mutant progeny have thesame functionality as that of the originally transformed cell.

[0116] Host cells which may be used in the expression systems of thepresent invention are not strictly limited, provided that they aresuitable for use in the expression of the EBI 1, 2, or 3 peptide ofinterest. Suitable hosts may often include eukaryotic cells.

[0117] Preferred eukaryotic hosts include, for example, yeast, fungi,insect cells, m alian cells either in vivo, or in tissue culture.Mammalian cells which may be useful as hosts include HeLa cells, cellsof fibroblast origin such as VERO or CHO-K1, or cells of lymphoidorigin, such as the hybridoma SP2/O-AG14 or the myeloma P3x63Sg8, andtheir derivatives. Preferred mammalian host cells include SP2/0 andJ558L, as well as neuroblastoma cell lines such as IMR 332 that mayprovide better capacities for correct post-translational processing.

[0118] In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.

[0119] Another preferred host is an insect cell, for example theDrosophila larvae. Using insect cells as hosts, the Drosophila alcoholdehydrogenase promoter can be used. Rubin, Science 240:1453-1459 (1988).Alternatively, baculovirus vectors can be engineered to express largeamounts of EBI 1, EBI 2, or EBI 3 in insect cells (Jasny, Science238:1653 (1987); Miller et al., In: Genetic Engineering (1986), Setlow,J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

[0120] Any of a series of yeast gene sequence expression systems can beutilized which incorporate promoter and termination elements from theactively expressed gene sequences coding for glycolytic enzymes areproduced in large quantities when yeast are grown in mediums rich inglucose. Know glycolytic gene sequences can also provide very efficienttranscriptional control signals.

[0121] Yeast provides substantial advantages in that it can also carryout post-translational peptide modifications. A number of recombinantDNA strategies exist which utilize strong promoter sequences and highcopy number of plasmids which can be utilized for production of thedesired proteins in yeast. Yeast recognizes leader sequences on clonedmammalian gene sequence products and secretes peptides bearing leadersequences (i.e., pre-peptides). For a mammalian host, several possiblevector systems are available for the expression of EBI 1, EBI 2, or EBI3.

[0122] A wide variety of transcriptional and translational regulatorysequences may be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus, simian virus,or the like, where the regulatory signals are associated with aparticular gene sequence which has a high level of expression.Alternatively, promoters from mammalian expression products, such asactin, collagen, myosin, and the like, may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the gene sequences can bemodulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

[0123] As discussed above, expression of EBI 1, EBI 2, or EBI 3 ineukaryotic hosts requires the use of eukaryotic regulatory regions. Suchregions will, in general, include a promoter region sufficient to directthe initiation of RNA synthesis. Preferred eukaryotic promoters include,for example, the promoter of the mouse metallothionein I gene sequence(Hamer et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter ofHerpes virus (McKnight, Cell 31:355-365 (1982)); the SV40 early promoter(Benoist et al., Nature (London) 290:304-310 (1981)); the yeast gal4gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA)79:6971-6975 (1982); Silver et al., Proc. Natl. Acad. Sci. (USA)81:5951-5955 (1984)).

[0124] As is widely known, translation of eukaryotic mRNA is initiatedat the codon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes EBI 1, EBI 2, or EBI 3 (or a functionalderivative thereof) does not contain any intervening codons which arecapable of encoding a methionine (i.e., AUG). The presence of suchcodons results either in a formation of a fusion protein (if the AUGcodon is in the same reading frame as the EBI 1, EBI 2, or EBI 3 codingsequence) or a frame-shift mutation (if the AUG codon is not in the samereading frame as the EBI 1, EBI 2, or EBI 3 coding sequence).

[0125] An EBI 1, EBI 2, or EBI 3 DNA segment and an operably linkedpromoter may be introduced into a recipient prokaryotic or eukaryoticcell either as a non-replicating DNA (or RNA) molecule, which may eitherbe a linear molecule or, more preferably, a closed covalent circularmolecule. Since such molecules are incapable of autonomous replication,the expression of the gene may occur through the transient expression ofthe introduced sequence. Alternatively, permanent expression may occurthrough the integration of the introduced DNA sequence into the hostchromosome.

[0126] In one embodiment, a vector is employed which is capable ofintegrating the desired gene sequences into the host cell chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes can be selected by also introducing one or more markerswhich allow for selection of host cells which contain the expressionvector. The marker may provide for prototrophy to an auxotropic host,biocide resistance, e.g. antibiotics, or heavy metals, such as copper,or the like. The selectable marker gene sequence can either be directlylinked to the DNA gene sequences to be expressed, or introduced into thesame cell by co-transfection. Additional elements may also be needed foroptimal synthesis of single chain binding protein mRNA. These elementsmay include splice signals, as well as transcription promoters,enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama, Molec.Cell. Biol. 0.3:280 (1983).

[0127] In a preferred embodiment, the introduced sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. Factors of importance in selecting aparticular plasmid or viral vector include: the ease with whichrecipient cells that contain the vector may be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to “shuttle” the vector between hostcells of different species. Preferred prokaryotic vectors includeplasmids such as those capable of replication in E. coli (such as, forexample, pBR322, ColE1, pSC101, pACYC 184, πVX. Such plasmids are, forexample, disclosed by Sambrook (cf. Molecular Cloning: A LaboratoryManual, second edition, edited by Sambrook, Fritsch & Maniatis, ColdSpring Harbor Laboratory, 1989)). Bacillus plasmids include pCl94,pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In:The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp.307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall et al.,J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophagessuch as φC31 (Chater et al., In: Sixth International Symposium onActinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp.45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect.Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742(1978)).

[0128] Preferred eukaryotic plasmids include, for example, BPV,vaccinia, SV40, 2-micron circle, and the like, or their derivatives.Such plasmids are well known in the art (Botstein et al., Miami Wntr.Symp. 19:265-274 (1982); Broach, In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, Cell28:203-204 (1982); Bollon et al., J. Clin. Hematol. Oncol. 10:39-48(1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3,Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980)).

[0129] Once the vector or DNA sequence containing the construct(s) hasbeen prepared for expression, the DNA construct(s) may be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,and the like. After the introduction of the vector, recipient cells aregrown in a selective medium, which selects for the growth ofvector-containing cells. Expression of the cloned gene sequence(s)results in the produciton of EBI 1, EBI 2, or EBI 3, or fragmentsthereof. This can take place in the transformed cells as such, orfollowing the induction of these cells to differentiate (for example, byadministration of bromodeoxyuracil to neuroblastoma cells or the like).

[0130] A variety of incubation conditions can be used to form thepeptide of the present invention. The most preferred conditions arethose which mimic physiological conditions.

[0131] G. An Antibody Having Binding Affinity to an EBI 1, EBI 2, andEBI 3 Polypeptide, or a Binding Fragment Thereof and a HybridomaContaining the Antibody.

[0132] In another embodiment, the present invention relates to anantiobdy having binding affinity to a polypeptide having an amino acidsequence selected from the group consisting of EBI 1, EBI 2, and EBI 3polypeptides, or a binding fragment thereof. In a preferred embodiment,the polypeptide has an amino acid sequence selected from the group ofsequences set forth in SEQ ID NO:2, SEQ ID NO: 4, and SEQ ID NO:6, ormutant or species variation thereof, or at least 7 contiguous aminoacids thereof (preferably, at least 10, 15, 20, or 30 contiguous aminoacids thereof). In another preferred embodiment, the antibody is amonoclonal antibody.

[0133] In another embodiment, the present invention relates to ahybridoma which produces the above-described monoclonal antibody, orbinding fragment thereof.

[0134] The EBI 1, EBI 2, or EBI 3 proteins of the present invention canbe used in a variety of procedures and methods, such as for thegeneration of antibodies, for use in identifying pharmaceuticalcompositions, and for studying DNA/protein interaction.

[0135] The EBI 1, EBI 2, or EBI 3 peptide of the present invention canbe used to produce antibodies or hybridomas. One skilled in the art willrecognize that if an antibody is desired, such a peptide would begenerated as described herein and used as an immunogen.

[0136] The antibodies of the present invention include monoclonal andpolyclonal antibodies, as well as fragments of these antibodies, andhumanized forms. Humanized forms of the antibodies of the presentinvention may be generated using one of the procedures known in the artsuch as chimerization or CDR grafting.

[0137] The invention also provides hybridomas which are capable ofproducing the above-described antibodies. A hybridoma is an immortalizedcell line which is capable of secreting a specific monoclonal antibody.

[0138] In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and Molecular Biology,Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St.Groth et al., J. Immunol. Methods 35:1-21 (1980)).

[0139] Any animal (mouse, rabbit, and the like) which is known toproduce antibodies can be immunized with the selected polypeptide.Methods for immunization are well known in the art. Such methods includesubcutaneous or interperitoneal injection of the polypeptide. Oneskilled in the art will recognize that the amount of polypeptide usedfor immunization will vary based on the animal which is immunized, theantigenicity of the polypeptide and the site of injection.

[0140] The polypeptide may be modified or administered in an adjuvant inorder to increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

[0141] For monoclonal antibodies, spleen cells from the immunizedanimals are removed, fused with myeloma cells, such as SP2/0-Ag14myeloma cells, and allowed to become monoclonal antibody producinghybridoma cells.

[0142] Any one of a number of methods well known in the art can be usedto identify the hybridoma cell which produces an antibody with thedesired characteristics. These include screening the hybridomas with anELISA assay, western blot analysis, or radioimmunoassay (Lutz et al.,Exp. Cell Res. 175:109-124 (1988)).

[0143] Hybridomas secreting the desired antibodies are cloned and theclass and subclass is determined using procedures known in the art(Campbell, Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, supra (1984)).

[0144] For polyclonal antibodies, antibody containing antisera isisolated from the immunized animal and is screened for the presence ofantibodies with the desired specificity using one of the above-describedprocedures.

[0145] In another embodiment of the present invention, theabove-described antibodies are detectably labeled. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluroescentlabels (such as FITC or rhodamine, and the like), paramagnetic atoms,and the like. Procedures for accomplishing such labeling are well knownin the art, for example, see (Stemberger et al., J. Histochem. Cytochem.18:315 (1970); Bayer et al., Meth. Enzym. 62:308 (1979); Engval et al.,Immunol. 109:129 (1972); Goding, J. Immunol. Meth. 13:215 (1976)). Thelabeled antibodies of the present invention can be used for in vitro, invivo, and in situ assays to identify cells or tissues which express aspecific peptide.

[0146] In another embodiment of the present invention, theabove-described antibodies are immobilized on a solid support. Examplesof such solid supports include plastics such as polycarbonate, complexcarbohydrates, such as agarose and sepharose, acrylic resins, such aspolyacrylamide and latex beads. Techniques for coupling antibodies tosuch solid supports are well known in the art (Weir et al., Handbook ofExperimental Immunology, 4th Ed., Blackwell Scientific Publications,Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34Academic Press, N.Y. (1974)). The immobilized antibodies of the presentinvention can be used for in vitro, in vivo, and in situ assays as wellas in immunochromatography.

[0147] Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,“Application of Synthetic Peptides: Antisense Peptides,” In SyntheticPeptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992), andKaspczak et al., Biochemistry 28:9230-8 (1989).

[0148] Anti-peptide peptides can be generated in one of two fashions.First, the anti-peptide peptides can be generated by replacing the basicamino acid residues found in the EBI 1, EBI 2, or EBI 3 peptide sequencewith acidic residues, while maintaining hydrophobic and uncharged polargroups. For example, lysine, arginine, and/or histidine residues arereplaced with aspartic acid or glutamic acid and glutamic acid residuesare replaced by lysine, arginine or histidine.

[0149] H. A Method of Detecting an EBI 1, EBI 2, or EBI 3 Polypeptide ina Sample.

[0150] In another embodiment, the present invention relates to a methodof detecting a polypeptide selected from the group consisting of EBI 1,EBI 2, EBI 3 in a sample, comprising: (a) contacting the sample with anabove-described antibody, under conditions such that immunocomplexesform, and (b) detecting the presence of said antibody bound to thepolypeptide. In detail, the methods comprise incubating a test samplewith one or more of the antibodies of the present invention and assayingwhether the antibody binds to the test sample. The presence of an EBI 1,EBI 2, or EBI 3 polypeptide or fragment thereof in a sample may indicatethe presence or infection of Epstein Barr Virus.

[0151] Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard, An Introduction to Radioimmunoassay andRelated Techniques, Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla., Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).

[0152] The immunological assay test samples of the present inventioninclude cells, protein or membrane extracts of cells, or biologicalfluids such as blood, serum, plasma, or urine. The test sample used inthe above-described method will vary based on the assay format, natureof the detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily adaptedin order to obtain a sample which is capable with the system utilized.

[0153] I. A Diagnostic Kit Comprising Antibodies to EBI 1, EBI 2, andEBI 3.

[0154] In another embodiment of the present invention, a kit is providedwhich contains all the necessary reagents to carry out the previouslydescribed methods of detection. The kit may comprise: (i) a firstcontainer means containing an above-described antibody; and (ii) secondcontainer means containing a conjugate comprising a binding partner ofthe antibody and a label. In another preferred embodiment, the kitfurther comprises one or more other containers comprising one or more ofthe following: wash reagents and reagents capable of detecting thepresence of bound antibodies. Examples of detection reagents include,but are not limited to, labeled secondary antibodies, or in thealternative, if the primary antibody is labeled, the chromophoric,enzymatic, or antibody binding reagents which are capable of reactingwith the labeled antibody. The compartmentalized kit may be as describedabove for nucleic acid probe kits.

[0155] One skilled in the art will readily recognize that the antibodiesdescribed in the present invention can readily be incorporated into oneof the established kit formats which are well known in the art.

[0156] The present invention is described in further detail in thefollowing non-limiting examples.

EXAMPLES

[0157] The following protocols and experimental details are referencedin the examples that follow:

[0158] Cells and cell lines. BL41 and BL30 are EBV(−) Burkitt lymphomacell lines. The BL41/B95-8 and BL41/P3HR1 cell lines were derived byinfecting BL41 with the transforming EBV strain, B95-8, or with thenon-transforming strain, P3HR1, respectively (Favrot, M. C., et al.,Intl. J. Cancer 38(6):901-6 (1986)). IB4 is a latently infected Blymphoblastoid cell line established by infection of lymphocytes withEBV (B95-8) in vitro. RHEK-1 (generous gift from Dr. Jong Rhim, NationalCancer Institute, Bethesda, Md.) is a human keratinocyte line derived byinfection of primary foreskin epithelial cells with an adenovirus12/SV40 hybrid-virus. K562 is a Philadelphia chromosome-positive humanchronic myeloid leukemia cell line. U937 is a histiocytic lymphoma cellline with monocytic features. HL60 is a promyelocytic leukemia line.HSB-2 and Jurkat are human T lymphoblastic leukemia cell lines. TK143was derived from a human osteosarcoma.

[0159] Human mononuclear cells (PBMC) were purified from peripheralblood by centrifugation on a ficoll cushion (Ficoll-Hypaque, Pharmacia,Vineland, N.J.). Cells were resuspended at 1×10⁶ cells/ml in RPMI mediumsupplemented with 20% fetal bovine serum, and were divided into parallelcultures grown 72 h with or without 2.5 μg/ml pokeweed mitogen (PWM,Sigma, St. Louis, Mo.). T cells were isolated from purified PBMCs byrosetting overnight with aminoethylisothiouronium bromide (AET) treatedsheep erythrocytes at 4° C., followed by centrifugation over ficoll.Pelleted erythrocytes were lysed with ammonium chloride. The remaining Tcells were resuspended in RPMI with 20% fetal bovine serum at 1×10⁶cells/ml. Phytohemagglutinin (PHA, Sigma) was added to a finalconcentration of 1.0 μg/ml. Cells were cultured for 72 h and harvestedfor extraction of total cellular RNA.

[0160] RNA preparation and analysis. Cytoplasmic RNA was isolated fromexponentially growing cells by a modification of the acidphenol/guanidinium isothiocyanate extraction procedure, followed byreprecipitation in guanidinium hydrochloride/ethanol. Total cellular RNAwas extracted from 0.2 to 2 g samples of human spleen and tonsilobtained from surgical specimens, and from human postmortem bone marrow.Tissues were homogenized in acid phenol/guanidinium isothiocyanate usinga rotary tissue homogenizer, extracted and precipitated. Afterdissolution in guanidinium hydrochloride and reprecipitation withethanol, human tissue RNA samples were resuspended in H₂O andprecipitated by addition of an equal volume of 8 M LiCl. Thepolyadenylated fractions of BL41 or BL41/B95-8 RNA were purified by 2successive cycles of chromatography on oligodeoxythymidylate cellulose.Polyadenylated 134 RNA was purified by a single round ofoligodeoxythymidylate selection. RNA samples (12 μg per lane) were sizefractionated on 0.66 M formaldehyde, 1% agarose gels and transferred tocharged nylon membranes (GeneScreen Plus, New England Nuclear,Billerica, Mass.) for subsequent hybridization analysis. To examine geneexpression in other human tissues, a commercially prepared blot waspurchased containing 2 μg of polyadenylated heart, brain, placenta,lung, liver, kidney, skeletal muscle and pancreas RNA (Multiple TissueNorthern, Clontech, Palo Alto, Calif.).

[0161] Probes were prepared from cloned cDNA inserts using randomhexamer primers and ³²P-dCTP. The beta actin probe was generated using apreviously described 1.4 kb cDNA (Alfieri, C., et al., Virology181(2):595-608 (1991)). The glyceraldehyde phosphate dehydrogenase(GAPDH) probe was prepared from a commercially obtained DNA fragment(Clontech). Filters were hybridized for 18 to 24 h at 47° C. in ahybridization buffer consisting of 50% formamide, 6×SSPE (20×SSPE: 3.0 MNaCl, 200 mM NaPO4, pH7.4, 20 mM EDTA), 1% SDS, 1× Denhardt's solution(100× Denhardt's: 2% BSA, 2% polyvinylpyrrolidone, 2% Ficoll), and 100μg/ml sheared single-stranded herring testis DNA. Filters were washedaccording to the manufacturer's instructions, with high stringencywashes performed at 67° C.-70° C. in 1% SDS, 0.2×SCC, and exposed topreflashed film (X-OMAT AR, Kodak, Rochester, N.Y.) at −80° C. for 2 hto 10 days. Autoradiographic signal intensities were quantitated bydensitometric scanning using a Beckman DU-8 spectrophotometer equippedwith a slab gel Compuset Module. Induction factors were calculated foreach probe as signal intensity ratios for EBV(+) versus EBV(−) cells,divided by the ratio of beta actin signal intensities.

[0162] cDNA library preparation. First strand cDNA was prepared from 5μg polyadenylylated BL41/B95-8 RNA using Moloney murine leukemia virusreverse transcriptase (SuperScript, Bethesda Research Laboratories,Gaithersburg, Md.) and oligodeoxthymidylate primers in a 100 μLreaction. Second strand cDNA was synthesized using E. coli DNApolymerase I and RNAse H. The double stranded cDNA was blunt-ended withT4 DNA polymerase and EcoRI methylated. After ligation of EcoRI linkers,the cDNA was EcoRI restriction digested and size fractionated by gelfiltration chromatography on Sepharose CL-4B. The purified cDNA wasligated to phosphorylated lambda gt10 arms (Promega, Madison, Wis.) andpackaged (Gigapack Gold, Stratagene, La Jolla, Calif.).

[0163] Subtractive probe preparation. Radiolabelled cDNA was preparedfrom 6 μg of polyadenylylated BL41 or BL41/B95-8 RNA in a 200 μLreaction containing 50 μg/ml random DNA hexamers; 0.5 mM dATP, dGTP,dTTP; 25 μM unlabelled dCTP; 1.0 mCi ³²P_dCTP (800 Ci/mMole, New EnglandNuclear); 2000 units recombinant Moloney murine leukemia virus reversetranscriptase. Reaction were 42° C. for 1 h. After precipitation,reaction products were resuspended in 0.1 M NaOH and incubated 20 min.at 65° C. to hydrolyze RNA templates. Probes were neutralized with 0.1 Macetic acid and size fractionated on G-50 Sephadex. Biotinylated RNA wasprepared from polyadenylylated BL41 RNA using a photoactivatableazido-aryl biotin reagent (Photoprobe Biotin, Vector Laboratories,Burlingame, Calif.) following the manufacturer's protocol. Probefractions were combined with 48 μg (for BL41/B95-8 probe) or 12 μg (forBL41 probe) biotinylated BL41 RNA and precipitated with ethanol.BL41/B95-8 probes were hydridized with an 8 fold excess (2 mg/ml) ofbiotinylated BL41 RNA; while BL41 control probes were hydridized with a2 fold excess (0.5 μg/ml) of biotinylated BL41 RNA. Hybridizations andsubtractions were performed using the “Subtractor” kit (Invitrogen, SanDiego, Calif.) according to the manufacturer's instructions. Theprecipitated cDNA/RNA mixtures were resuspended in 10 to 20 μL H₂O andheated to 100° C. for 1 min. An equal volume of 2× hybridization buffer(Invitrogen) was added and the mixture was incubated at 65° C. for 20 to24 h. Following addition of an equal volume of HEPES buffer (10 mMHEPES, pH 7.5, mM EDTA), 20 μg streptavidin was added and the mixturewas incubated on ice for 10 min. Biotinylated RNA and RNA:cDNA duplexes,complexed with avidin, were removed by repeated phenol/chloroformextractions. The single stranded, subtracted BL41 cDNA probe whichremained in the aqueous phase was used directly for in situ filterhybridizations. Aqueous phase BL41/B95-8 cDNA probe was precipitatedwith ethanol and subjected to a second round of subtraction underidentical conditions prior to use in filter hydridizations. Duplicatefilters were made from 145 mm plates containing 6000 recombinantbacteriophage and were hybridized in parallel to equal amounts ofBL41/B95-8 or BL41 subtracted probes. Filters were hybridized at 48° C.for 48 to 72 h in a buffer consisting of 50% formamide, 6×SSPE, 1% SDS,10% dextran sulfate, 2× Denhardt's solution, 100 μg/ml shearedsingle-stranded herring testis DNA, and 10 μg/ml poly rA:rU (Sigma, St.Louis, Mo.). Filters were washed at 72° C. in 0.2×SSC and exposed 3 to 7days to preflashed film (Kodak X-OMAT AR). Differentially expressedgenes were identified by overlaying films from corresponding filters.Clones selected on primary screening were rescreened once at low densityto verify differential expression and for plaque purification.

[0164] Analysis of clones. DNA was extracted from bulk liquid culturesof purified lambda gt10 clones and digested with EcoRI. cDNA insertswere purified by agarose gel electrophoresis and subcloned withpBluescript (+). Nucleotide sequences were determined and were comparedby the BLAST algorithm (Altschul, S. F., et al., J. Mol. Biol.215(3):403-10 (1990)) with known sequences resident in the NationalCenter for Biotechnology Information databases using the ExperimentalGENINFO® BLAST Network Service, accessed through the Molecular BiologyComputer Research Resource of the Dana-Faber Cancer Institute. Multiplesequence alignments were performed by the method of Higgins and Sharp(Higgins and Sharp, Gene 73(1):237-44 (1988)) using the CLUSTAL program(PC Gene, IntelliGenetics, Mountain View, Calif.) with open gap and unitgap costs of 10.

EXAMPLE I Identification of cDNA Clones of EB V Induced RNAs bySubtracted Probe Hybridization

[0165] CDNA clones of RNA from an in vitro EBV-infected BL cell line,BL41/B95-8 [EBV(+) BL41], were differentially screened with an EBV(+)BL41 cDNA probe from which sequences complementary to EBV(−)BL41 cellRNA had been specifically removed, and with an EBV(−) BL41 control cDNAprobe. Sequences complimentary to EBV(−) BL41 RNA were removed from theEBV (+) BL41 RNA cDNA probes by two subtractions with an 8 fold excessof biotinylated EBV(−) BL41 RNA. Overall, 85-95% of the labeled EBV(+)BL41 probe was removed by the two subtractions. EBV(−) BL41 cDNA controlprobe was subtracted only once, removing 60-85% of the probe; therebyreducing hybridization to plaques containing cDNAs from abundant RNAs sothat hybridization to cDNAs from less abundant BL41 RNAs was evident.

[0166] Seventy-five phage cDNA clones differentially hybridized to theEBV(+) BL41 probe on the first screen of 75,000 recombinant phage.Twenty-five clones were consistently positive on rescreening. Theeighteen clones which demonstrated the greatest reactivity with theEBV(+) versus the EBV(−) BL41 cDNA probes were selected for nucleotidesequencing and RNA blot hybridization.

EXAMPLE 2 Nucleotide Sequences of EB V Induced cDNAs

[0167] The first 12 clones are described in Table 1. Ten clones matched7 previously characterized genes: two independent clones each of thecomplement receptor type 2 (CD21), the serglycin proteoglycan coreprotein and vimentin; and one clone each of cathepsin H, annexin VI(p68), the myristylated alanine-rich protein kinase C substrate (MARCKS)and the lymphocyte hyaluronic acid receptor (CD44). The 2.6 kb MARCKScDNA precisely matched the previous 1.58 kb human MARCKS cDNA clone(Harlan, D. M., et al. J. Biol. Chem. 266(22):14399-405 (1991)) at its 5prime end. The 3 prime untranslated region of the new clone is highlyhomologous to bovine MARCKS cDNA (Stumpo, D. J., et al., Proc. Natl.Acad. Sci. USA 86(11):4012-6(1989)).

[0168] The two remaining clones from novel RNAs, EBV induced genes 1(EBI 1) and 2 (EBI 2), whose nucleotide sequences can be predicted toencode G-protein coupled peptide receptors. The complete nucleotide anddeduced amino acid sequences of the EBI 1 and EBI 2 cDNAs are shown inFIGS. 1A and 1D, respectively. Because the first EBI 1 cDNA was 1.2.kb,significantly shorter than the 2.4 kb RNA, 20 other cDNA clones wereobtained using the initial cDNA as a probe. The largest clone is 2153nucleotides (nt) and has a 1134 nt open-reading frame (FIG. 1A). Thisclone is probably nearly full length, since it is close to the expectedsize, considering it has only a short poly A tail. Translation is likelyto initiate from either of two AUGs, at nt 64-66 or 82-84, the first ofwhich conforms to a consensus translation initiation sequence (Kozak,M., J. Biol. Chem. 266(30):19867-70 (1991)). An in-frame stop codon atnt 10-12 is consistent with downstream initiation at nt 64-66. Thepolypeptide encoded by the sequence beginning at nt 64 has a predictedmolecular weight of 42.7 kD and includes eight hydrophobic domainslikely to mediate membrane insertion. The first hydrophobic domainbegins at the amino terminus and ends at a predicted signal peptidasecleavage site. The 7 remaining hydrophobic domains are characteristic ofthe G-protein coupled receptor family. Potential asparagine linkedglycosylation sites are present in the extracellular amino terminalsegment and in the third extracellular loop.

[0169] Since the initial EBI 2 cDNA was 1643 nt and approximated thesize expected from a 1.9 kb polyadenylated RNA, further cDNA clones werenot obtained. The EBI 2 cDNA contains a 1083 nt open reading frame withtwo methionine codons are at nt 34-36 and 46-48 (FIG. 1B). Althoughneither methionine codon is in a favored initation context (Kozak, M.,J. Biol. Chem. 266(30):19867-70 (1991)), an upstream, in-frametermination codon and the absence of other potential open reading framesis consistent with translation initiating at the first or secondmethionine codon. Initiation at the first would result in a 41.2 kDprotein. The deduced amino acid sequence predicts 7 hydrophobictransmembrane segments in the characteristic configuration of G-proteincoupled receptors. In contrast to the EBI 1 protein, EBI 2 lacks asignal pepetide. A possible N-linked glycosylation site is found in theamino terminal extracellular domain. Though the EBI 2 cDNA lacks apolyadenylate tail, a canonical polyadenylation signal (AATAAA) near the3 prime end is consistent with the cDNA being essentially complete.

EXAMPLE 3 Comparison of EBI 1 and 2 with Other G Protein CoupledReceptors

[0170] The EBI 1 and EBI 2 nucleotide and predicted amino acid sequenceswere compared with the Genbank (release 72 and updates), EMBL (release31), Genbank translation, Swiss protein (release 22) and ProteinIdentification Resource (PIR, release 33) databases, using the BLASTalgorithm (Alschul, S. F., et al., J. Mol. Biol. 213(3):403-10 (1990)).EBI 1 and EBI 2 are homologous to G protein associated receptors. EBI 1is highly homologous to the human high or low affinity interleukin 8(IL-8) receptors at both the nucleotide (data not shown) and amino acidsequence levels. IL8 receptor itself is not expressed on lymphocytes(Holmes, W. E., et al., Science 253(5025):1278-80 (1991)); Murphy andTiffany, et al., Science 253:1280-1283 (1991). Excluding the putativeEBI 1 signal peptide, the overall amino acid identity among the 3proteins exceeds 30%, with conservative changes observed at many of thenon-identical residues. The identity increases to 40% when EBI 1 iscompared with either IL-8 receptor individually. Additional similaritieswith the IL-8 receptors include a high proportion of serine andthreonine near the carboxy terminus, and a highly acidic amino terminalextracellular domain. The IL-8 receptor acidic residues are implicatedin binding IL-8 basic amino acids (Holmes, W. E., et al., Science253(5025):1278-80 (1991); Murphy and Tiffany, et al., Science253:1280-1283 (1991)).

[0171] The EBI 2 gene does not have such a close homologue. EBI 2 has24% amino acid indentity to the thrombin receptor (Vu, T. K., et al.,Cell 64(6):1057-68 (1991)). Less extensive homologies are observed witha number of other G-protein coupled receptors, including the receptorsfor vasoactive intestinal polypeptide, somatostatin (type 1) andangiotensin II, as well as the low affinity IL-8 receptor. EBI 2 alsoexhibits more distant homologies with EBI 1 and the high affinity IL-8receptor. Significantly, these are the same proteins which, in differentorder, exhibit the closest homologies with the EBI 1 protein. Togetherthey constitute a subfamily of G-protein coupled peptide receptors. Thegreatest conservation of residues among these proteins extends from thefirst transmembrane domain to the second intracellular loop. Because ofthe particular conservation of an amino acid sequence among these Gprotein coupled receptors, we are able to identify a new highlyconserved sequence motif at the carboxy end of TM III and the adjacentsecond intracellular loop. This motif, S-(I/L)-D-R-(Y/F)-X-X-X-X, with Xbeing a hydrophobic amino acid, is in a wide variety of G-proteincoupled receptors; and is not in other proteins in the data basessurveyed. Other highly conserved features of G protein coupled receptorsin EBI 1 and 2 include the asparagine in TM I, the proline in TM II, theaspartate in the first intracellular loop, and the tryptophane andcysteine in the first extracellular loop. This cysteine has beenpostulated to be involved in disulfide linkage to a conserved cysteinepresent in the second extracellular loop in several other receptors,including the beta adrenergic and thrombin receptors.

EXAMPLE 4 Analysis of Induced Gene Expression by RNA Blot Hybridization

[0172] Probes from seven of the nine EBV induced cDNAs were hybridizedto identical blots of polyadenylated RNA from the EBV(+) or EBV(−) BL41cell lines or from the EBV transformed lymphoblastoid cell line, IB4(FIG. 2). Vimentin and CD21 were previously shown to be EBV induced andwere not further evaluated. The RNAs loaded in the EBV(+), BL41. andEBV(−) BL41 lanes were standardized with respect to beta actinreactivity. Significantly less IB4 cell RNA was used due to the highabundance of the putative induced gene RNAs in these cells (FIG. 2,Actin probe). Probes from each of the cDNA clones detected RNAs whichare significantly more abundant in both IB4 and EBV(+) BL41 cells thanin EBV(−) BL41 cells. Induction factors indicated in Table 1 weredetermined by quantitative densitometric scanning of autoradiographs andreflect the fold enhancement of signal intensities in EBV(+) BL41 cellscompared with EBV(−) BL41 cells, corrected for the ratio of actinreactivities. Standardization by actin reactivity, however,significantly underestimates the absolute induction levels since actinis induced 3-fold by EBV infection of BL41 cells relative toglyceraldehyde phosphate dehydrogenase, (GAPDH), or to total RNA amountsquantitated spectrophotmetrically. To achieve equal actin signalintensitites, 3-fold more EBV(−) BL41 than EBV(+) RNA was loaded perlane. Importantly, each of the RNAs was at least as abundant in IB4cells relative to GADPH as in EBV(+) BL41 (FIG. 2).

[0173] EBI 1, EBI 2, CD44 and MARCKS are the most induced of the sevengenes. The CD44 gene encodes three distinct RNAs of 1.6, 2.2 and 4.8 kbrespectively in both IB4 and EBV(+) BL41 cells. No CD44 RNA was detectedEBV(−) BL41 cells even after prolonged autoradiographic exposures. EBI 2RNA was also undetectable in EBV(−) BL41 cells.

EXAMPLE 5 Expression of EBI 1 and 2 in Human Cell Lines and Tissues

[0174] The expression of EBI 1 and 2 in human cell lines and tissues wasevaluated by hybridizing actin, EBI 1 or EBI 2 probes to blots of cellline or tissue RNAs. While EBI 1 is weakly expressed in BL41, EBI 2 isnot; and, neither EBI 1 nor EBI 2 are expressed in another EBV(−) BLcell line, BL30 (FIG. 3). EBI 1 and EBI 2 RNAs are abundant in primaryhuman lymphocytes transformed by EBV in vitro and propagated ascontinuous lymphoblastoid cell lines for several years (IB4) or for lessthan 1 year (W91-LCL) (FIG. 3). EBI 1 RNA is faintly detectable in thehuman T cell line Jurkat, and is abundantly expressed in a second T cellline, HSB-2 (FIG. 3). EBI 2 RNA is not detected in either T cell line(FIG. 3), nor in a third T cell line, MOLT-4. EBI 1 is not expressed inthe human promyelocytic line, HL 60, the chronic myelogeneous leukemiacell line K562, the epithelial cell line, RHEK-1, the fibroblast-likeosteosarcoma cell line, TK143, or the monocytic cell line, U937 (FIG.3). EBI 2, however, is expressed weakly, relative to actin, in HL60,U937, (U937 RNA is partially degraded) or HeLa cells (FIG. 3).

[0175] EBI 1 and 2 RNAs are abundant in human spleen, somewhat lessabundant relative to actin in tonsil are not detectable in bone marrow(FIG. 3). Both genes were expressed in resting PBMCs at levelscomparable to IB4 or LCL-W91 B lyumphoblastoid cells (FIG. 3).Expression increased in parallel cultures stimulated for 72 h withpokeweed mitogen (PWM), although actin expression also increased withPWM (FIG. 3). The EBI 1 and 2 RNA in stimulated and non-stimulated PBMCcultures is likely to be mostly in B lymphocytes since EBI 1 RNA is atlow levels and EBI 2 RNA is absent from phytohemagglutinin stimulated,PBMC derived, T lymphocytes (FIG. 3). These findings are consistent withexpression patterns observed in T cell lines.

[0176] EBI 1 and 2 RNA levels were also evaluated in a variety ofnon-hematopoietic human tissues. The EBI 1 probe detects small amountsof RNA in both lung and pancreas (FIG. 4). Rehybridization of this blotwith an immunoglobulin mu chain probe (FIG. 4, Igu probe) indicated thatthese tissue preparations contained significant amounts ofimmunoglobulin RNA, probably due to B lymphocytes in the tissues. SinceEBI 1 RNA is abundant in peripheral blood lymphocytes, the EBI 1 RNA inthe lung and pancreas is likely to be due to B lymphocytes. Similarly,the low level of EBI 2 RNA detected in pancreatic tissue is probably dueto infiltrating B lymphocytes (FIG. 4). However, the abundance of EBI 2RNA in the lung is too great to attribute to lymphocyte contaminationand is more likely due to specific expression in pulmonary epithelialcells or macrophages (FIG. 4).

EXAMPLE 6 Cloning and Characterization of EBI 3

[0177] Subtractive hybridization screening of a BL41/B95-8 cDNA libraryhas permitted the identification of a number of genes expressed athigher levels in EBV-infected BL cells compared with matched EBV(−)cells. Twenty-five putative EBV-induced gene clones were initiallyisolated. Of these, 13 clones matched 8 previously known genes. Theremaining 12 clones represented 10 novel genes. Two of these clones werederived from transcripts of a previously uncharacterized gene designatedEBV-induced gene 3 (EBI 3).

[0178] The complete nucleotide and amino acid sequence of the larger EBI3 clone are shown in FIG. 5 (SEQ ID NO:5 and SEQ ID NO:6, respectively).The 1182 nucleotide cDNA contains a 690 nucleotide open reading frame. Aunique AUG codon preceding this reading frame at nucleotides 14-16conforms to the Kozak consensus translation initiation sequence.Initiation from this site results in the synthesis of a polypeptide witha predicted molecular mass of 25,380 Daltons. The first 20 amino acidsare highly hydrophobic and likely form a signal peptide for membranetranslocation with a predicted signal peptidase cleavage site followinga glycine residue at position 20. Two potential asparagine-linkedglycosylation sites are also identified. However, no other hydrophobicsegments capable of forming the transmembrane domain of an integralmembrane protein are evident. To verify the structure of this cDNA, fiveadditional clones were retrieved from the library. All of theseexhibited identical sequences throughout the putative carboxy-terminalportion of the predicted protein. The 3′ end of the EBI 3 nucleotidesequence is notable for its homology to the left monomer of the humanAlu repeat element. This homology extends to and includes the A-richsequences which immediately precede the polyadenylate tail of the mRNA.

[0179] The EBI 3 nucleotide and amino acid sequences were compared withall known sequences of Genbank nucleic acid, and Genbank translation,Protein Identification Resource (PIR) and Swiss Protein databases,respectively, using the Experimental GENINFO(R) BLAST-server network ofthe National Center for Biotechnology Information. No significantnucleotide homologies were observed, excluding matches with the 3′untranslated Alu repeat. However, the predicted EBI 3 protein isapproximately 30% identical to the receptor for ciliary neurotrophicfactor (CNTF), with conservative amino acid changes at many of thenon-identical residues. Of particular significance is the pattern ofconserved residues which include 4 cysteines at positions 35, 46, 80 and90 respectively of the complete EBI 3 protein sequence; tryptophanes atpositions 48 and 150; proline at position 125; and aliphatic hydrophobicresidues at positions 128, 136, 148 and 204. In addition, the EBI 3sequence LSDWS at residues 215 to 219 closely matches the WSDWS sequenceof the CNTF receptor. These conserved structural features arecharacteristic of and unique to members of the cytokine receptor family.The predicted EBI 3 protein exhibits less extensive homologies with thep40 subunit of interleukin 12 (IL-12), also known as natural killer cellstimulatory factor. Though a secreted protein, IL-12 p40 possesses thesame conserved residues and is also a member of the cytokine receptorfamily. In addition, the carboxy terminal 100 amino acids of the EBI 3protein exhibit structural homologies with type III fibronectin domainsof a variety of adhesion related molecules, including tenascin,cytotactin and the neural cell adhesion molecule, NCAM. This feature hasalso been described among other cytokine receptor family members.

[0180] Hybridization of a ³²P-labeled EBI 3 probe to RNA blots detects a1.5 kb RNA in the EBV-infected cell lines 1B4 and BL41/B95-8 (FIG. 6).EBI 3 RNA is undetectable, however, in the EVB(−) control cell lineBL41. To provide standards for the amounts of RNA loaded in each line,parallel blots were hybridized with probes for glyceraldehyde phosphatedehydrogenase (GAPDH) and actin. These probes indicate that the BL41lane contains as much or more RNA than the EBV-infected cell lanes.

[0181] Examination of a series of human cell lines and lymphoid tissuesindicated that EBI 3 is expressed at very low levels in normalunfractionated resting lymphocytes of spleen and tonsil, but isundetectable in peripheral blood mononuclear cells (PBMC). However,stimulation of PBMC with the B and T lymphocyte activating agent,pokeweed mitogen, results in induction of the EBI 3 mRNA. Lower levelsof EBI 3 RNA were detected in phytohemagglutinin stimulated peripheralblood T lymphocytes. In addition to IB4 and BL41/B95-8 cells, a recentlyestablished lymphoblastoid cell line transformed with the W91 EBV strainalso exhibited significant EBI 3 expression. EBI 3 RNA was undetectablein a second EBV(−) BL cell line, BL30, in BL41 cells infected with thenon-transforming P3HR1 EBV strain, and in all human myeloid, T lymphoidor epithelial cell line examined.

[0182] Expression of EBI 3 RNA was also analyzed in a variety ofnon-lymphoid human tissues (FIG. 7A). Abundant expression was observedin placenta, significantly exceeding expression levels observed in anylymphoid cell type. EBI 3 RNA was also faintly detectable in liver RNA.However, rehybridization of this blot with an immunoglobulin 1 heavychain probe indicated detectable Ig gene expression, probably due toinfiltration of liver tissue with lymphocytes in vivo. The apparentexpression of EBI 3 in liver could therefore be due to expression inresident lymphocytes.

[0183] All publications mentioned hereinabove are hereby incorporated intheir entirety by reference.

[0184] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims. TABLE 1 Summary of EBVInduced RNA: cDNA Clones Clone Gene cDNA Size (kb) RNA Size (kb)Induction¹ 1.1 CD44 1, 3 1.6, 2.2, 5.0 >100X 3.3; 7.3 CD21 2.1; 1.8 4.86.5 MARCKS 2.6 2.9  30X 8.2 Cathepsin H 1.5 1.7   6X 10.4; 11.4Serglycin 1.1; 1.1 1.4   3.5X 12.3  Annexin VI 2.3 3.0   5X 12.5; 13.0Vimentin 1.0; 1.8 2.0 6.4 EBI 1 1.2 (2.14)² 2.4  21X 3.2 EBI 2 1.641.9 >200X Beta actin 2.2   3X³

[0185]

1 7 1 2154 DNA Homo sapiens CDS 64..1197 1 ggaattccgt agtgcgaggccgggcacagc cttcctgtgt ggttttaccg cccagagagc 60 gtc atg gac ctg ggg aaacca atg aaa agc gtg ctg gtg gtg gct ctc 108 Met Asp Leu Gly Lys Pro MetLys Ser Val Leu Val Val Ala Leu 1 5 10 15 ctt gtc att ttc cag gta tgcctg tgt caa gat gag gtc acg gac gat 156 Leu Val Ile Phe Gln Val Cys LeuCys Gln Asp Glu Val Thr Asp Asp 20 25 30 tac atc gga gac aac acc aca gtggac tac act ttg ttc gag tct ttg 204 Tyr Ile Gly Asp Asn Thr Thr Val AspTyr Thr Leu Phe Glu Ser Leu 35 40 45 tgc tcc aag aag gac gtg cgg aac tttaaa gcc tgg ttc ctc cct atc 252 Cys Ser Lys Lys Asp Val Arg Asn Phe LysAla Trp Phe Leu Pro Ile 50 55 60 atg tac tcc atc att tgt ttc gtg ggc ctactg ggc aat ggg ctg gtc 300 Met Tyr Ser Ile Ile Cys Phe Val Gly Leu LeuGly Asn Gly Leu Val 65 70 75 gtg ttg acc tat atc tat ttc aag agg ctc aagacc atg acc gat acc 348 Val Leu Thr Tyr Ile Tyr Phe Lys Arg Leu Lys ThrMet Thr Asp Thr 80 85 90 95 tac ctg ctc aac ctg gcg gtg gca gac atc ctcttc ctc ctg acc ctt 396 Tyr Leu Leu Asn Leu Ala Val Ala Asp Ile Leu PheLeu Leu Thr Leu 100 105 110 ccc ttc tgg gcc tac agc gcg gcc aag tcc tgggtc ttc ggt gtc cac 444 Pro Phe Trp Ala Tyr Ser Ala Ala Lys Ser Trp ValPhe Gly Val His 115 120 125 ttt tgc aag ctc atc ttt gcc atc tac aag atgagc ttc ttc agt ggc 492 Phe Cys Lys Leu Ile Phe Ala Ile Tyr Lys Met SerPhe Phe Ser Gly 130 135 140 atg ctc cta ctt ctt tgc atc agc att gac cgctac gtg gcc atc gtc 540 Met Leu Leu Leu Leu Cys Ile Ser Ile Asp Arg TyrVal Ala Ile Val 145 150 155 cag gct gtc tca gct cac cgc cac cgt gcc cgcgtc ctt ctc atc agc 588 Gln Ala Val Ser Ala His Arg His Arg Ala Arg ValLeu Leu Ile Ser 160 165 170 175 aag ctg tcc tgt gtg ggc agc gcc ata ctagcc aca gtg ctc tcc atc 636 Lys Leu Ser Cys Val Gly Ser Ala Ile Leu AlaThr Val Leu Ser Ile 180 185 190 cca gag ctc ctg tac agt gac ctc cag aggagc agc agt gag caa gcg 684 Pro Glu Leu Leu Tyr Ser Asp Leu Gln Arg SerSer Ser Glu Gln Ala 195 200 205 atg cga tgc tct ctc atc aca gag cat gtggag gcc ttt atc acc atc 732 Met Arg Cys Ser Leu Ile Thr Glu His Val GluAla Phe Ile Thr Ile 210 215 220 cag gtg gcc cag atg gtg atc ggc ttt ctggtc ccc ctg ctg gcc atg 780 Gln Val Ala Gln Met Val Ile Gly Phe Leu ValPro Leu Leu Ala Met 225 230 235 agc ttc tgt tac ctt gtc atc atc cgc accctg ctc cag gca cgc aac 828 Ser Phe Cys Tyr Leu Val Ile Ile Arg Thr LeuLeu Gln Ala Arg Asn 240 245 250 255 ttt gag cgc aac aag gcc atc aag gtgatc atc gct gtg gtc gtg gtc 876 Phe Glu Arg Asn Lys Ala Ile Lys Val IleIle Ala Val Val Val Val 260 265 270 ttc ata gtc ttc cag ctg ccc tac aatggg gtg gtc ctg gcc cag acg 924 Phe Ile Val Phe Gln Leu Pro Tyr Asn GlyVal Val Leu Ala Gln Thr 275 280 285 gtg gcc aac ttc aac atc acc agt agcacc tgt gag ctc agt aag caa 972 Val Ala Asn Phe Asn Ile Thr Ser Ser ThrCys Glu Leu Ser Lys Gln 290 295 300 ctc aac atc gcc tac gac gtc acc tacagc ctg gcc tgc gtc cgc tgc 1020 Leu Asn Ile Ala Tyr Asp Val Thr Tyr SerLeu Ala Cys Val Arg Cys 305 310 315 tgc gtc aac cct ttc ttg tac gcc ttcatc ggc gtc aag ttc cgc aac 1068 Cys Val Asn Pro Phe Leu Tyr Ala Phe IleGly Val Lys Phe Arg Asn 320 325 330 335 gat atc ttc aag ctc ttc aag gacctg ggc tgc ctc agc cag gag cag 1116 Asp Ile Phe Lys Leu Phe Lys Asp LeuGly Cys Leu Ser Gln Glu Gln 340 345 350 ctc cgg cag tgg tct tcc tgt cggcac atc cgg cgc tcc tcc atg agt 1164 Leu Arg Gln Trp Ser Ser Cys Arg HisIle Arg Arg Ser Ser Met Ser 355 360 365 gtg gag gcc gag acc acc acc accttc tcc cca taggcgactc ttctgcctgg 1217 Val Glu Ala Glu Thr Thr Thr ThrPhe Ser Pro 370 375 actagaggga cctctcccag ggtccctggg gtggggatagggagcagatg caatgactca 1277 ggacatcccc ccgccaaaag ctgctcaggg gaaaaagcagctctcccctc agagtgcaag 1337 cccctgctcc agaagatagc ttcaccccaa tcccagctacctcaaccaat gccaaaaaaa 1397 gacagggctg ataagctaac accagacaga caacactgggaaacagaggc tattgtcccc 1457 taaaccaaaa actgaaagtg aaagtccaga aactgttcccacctgctgga gtgaaggggc 1517 caaggagggt gagtgcaagg ggcgtgggag tggcctgaagagtcctctga atgaaccttc 1577 tggcctccca cagactcaaa tgctcagacc agctcttccgaaaaccaggc cttatctcca 1637 agaccagaga tagtggggag acttcttggc ttggtgaggaaaagcggaca tcagctggtc 1697 aaacaaactc tctgaacccc tccctccatc gttttcttcactgtcctcca agccagcggg 1757 aatggcagct gccacgccgc cctaaaagca cactcatcccctcacttgcc gcgtcgccct 1817 cccaggctct caacagggga gagtgtggtg tttcctgcaggccaggccag ctgcctccgc 1877 gtgatcaaag ccacactctg ggctccagag tggggatgacatgcactcag ctcttggctc 1937 cactgggatg ggaggagagg acaagggaaa tgtcaggggcggggagggtg acagtggccg 1997 cccaaggcca cgagcttgtt ctttgttctt tgtcacagggactgaaaacc tctcctcatg 2057 ttctgctttc gattcgttaa gagagcaaca ttttacccacacacagataa agttttccct 2117 tgaggaaaca acagctttaa aaaaaaaaaa ggaattc 21542 378 PRT Homo sapiens 2 Met Asp Leu Gly Lys Pro Met Lys Ser Val Leu ValVal Ala Leu Leu 1 5 10 15 Val Ile Phe Gln Val Cys Leu Cys Gln Asp GluVal Thr Asp Asp Tyr 20 25 30 Ile Gly Asp Asn Thr Thr Val Asp Tyr Thr LeuPhe Glu Ser Leu Cys 35 40 45 Ser Lys Lys Asp Val Arg Asn Phe Lys Ala TrpPhe Leu Pro Ile Met 50 55 60 Tyr Ser Ile Ile Cys Phe Val Gly Leu Leu GlyAsn Gly Leu Val Val 65 70 75 80 Leu Thr Tyr Ile Tyr Phe Lys Arg Leu LysThr Met Thr Asp Thr Tyr 85 90 95 Leu Leu Asn Leu Ala Val Ala Asp Ile LeuPhe Leu Leu Thr Leu Pro 100 105 110 Phe Trp Ala Tyr Ser Ala Ala Lys SerTrp Val Phe Gly Val His Phe 115 120 125 Cys Lys Leu Ile Phe Ala Ile TyrLys Met Ser Phe Phe Ser Gly Met 130 135 140 Leu Leu Leu Leu Cys Ile SerIle Asp Arg Tyr Val Ala Ile Val Gln 145 150 155 160 Ala Val Ser Ala HisArg His Arg Ala Arg Val Leu Leu Ile Ser Lys 165 170 175 Leu Ser Cys ValGly Ser Ala Ile Leu Ala Thr Val Leu Ser Ile Pro 180 185 190 Glu Leu LeuTyr Ser Asp Leu Gln Arg Ser Ser Ser Glu Gln Ala Met 195 200 205 Arg CysSer Leu Ile Thr Glu His Val Glu Ala Phe Ile Thr Ile Gln 210 215 220 ValAla Gln Met Val Ile Gly Phe Leu Val Pro Leu Leu Ala Met Ser 225 230 235240 Phe Cys Tyr Leu Val Ile Ile Arg Thr Leu Leu Gln Ala Arg Asn Phe 245250 255 Glu Arg Asn Lys Ala Ile Lys Val Ile Ile Ala Val Val Val Val Phe260 265 270 Ile Val Phe Gln Leu Pro Tyr Asn Gly Val Val Leu Ala Gln ThrVal 275 280 285 Ala Asn Phe Asn Ile Thr Ser Ser Thr Cys Glu Leu Ser LysGln Leu 290 295 300 Asn Ile Ala Tyr Asp Val Thr Tyr Ser Leu Ala Cys ValArg Cys Cys 305 310 315 320 Val Asn Pro Phe Leu Tyr Ala Phe Ile Gly ValLys Phe Arg Asn Asp 325 330 335 Ile Phe Lys Leu Phe Lys Asp Leu Gly CysLeu Ser Gln Glu Gln Leu 340 345 350 Arg Gln Trp Ser Ser Cys Arg His IleArg Arg Ser Ser Met Ser Val 355 360 365 Glu Ala Glu Thr Thr Thr Thr PheSer Pro 370 375 3 1643 DNA Homo sapiens CDS 34..1116 3 ggaattccctgatatacacc tggaccacca cca atg gat ata caa atg gca aac 54 Met Asp Ile GlnMet Ala Asn 1 5 aat ttt act ccg ccc tct gca act cct cag gga aat gac tgtgac ctc 102 Asn Phe Thr Pro Pro Ser Ala Thr Pro Gln Gly Asn Asp Cys AspLeu 10 15 20 tat gca cat cac agc acg gcc agg ata gta atg cct ctg cat tacagc 150 Tyr Ala His His Ser Thr Ala Arg Ile Val Met Pro Leu His Tyr Ser25 30 35 ctc gtc ttc atc att ggg ctc gtg gga aac tta cta gcc ttg gtc gtc198 Leu Val Phe Ile Ile Gly Leu Val Gly Asn Leu Leu Ala Leu Val Val 4045 50 55 att gtt caa aac agg aaa aaa atc aac tct acc acc ctc tat tca aca246 Ile Val Gln Asn Arg Lys Lys Ile Asn Ser Thr Thr Leu Tyr Ser Thr 6065 70 aat ttg gtg att tct gat ata ctt ttt acc acg gct ttg cct aca cga294 Asn Leu Val Ile Ser Asp Ile Leu Phe Thr Thr Ala Leu Pro Thr Arg 7580 85 ata gcc tac tat gca atg ggc ttt gac tgg aga atc gga gat gcc ttg342 Ile Ala Tyr Tyr Ala Met Gly Phe Asp Trp Arg Ile Gly Asp Ala Leu 9095 100 tgt agg ata act gcg cta gtg ttt tac atc aac aca tat gca ggt gtg390 Cys Arg Ile Thr Ala Leu Val Phe Tyr Ile Asn Thr Tyr Ala Gly Val 105110 115 aac ttt atg acc tgc ctg agt att gac cgc ttc att gct gtg gtg cac438 Asn Phe Met Thr Cys Leu Ser Ile Asp Arg Phe Ile Ala Val Val His 120125 130 135 cct cta cgc tac aac aag ata aaa agg att gaa cat gca aaa ggcgtg 486 Pro Leu Arg Tyr Asn Lys Ile Lys Arg Ile Glu His Ala Lys Gly Val140 145 150 tgc ata ttt gtc tgg att cta gta ttt gct cag aca ctc cca ctcctc 534 Cys Ile Phe Val Trp Ile Leu Val Phe Ala Gln Thr Leu Pro Leu Leu155 160 165 atc aac cct atg tca aag cag gag gct gaa agg att aca tgc atggag 582 Ile Asn Pro Met Ser Lys Gln Glu Ala Glu Arg Ile Thr Cys Met Glu170 175 180 tat cca aac ttt gaa gaa act aaa tct ctt ccc tgg att ctg cttggg 630 Tyr Pro Asn Phe Glu Glu Thr Lys Ser Leu Pro Trp Ile Leu Leu Gly185 190 195 gca tgt ttc ata gga tat gta ctt cca ctt ata atc att ctc atctgc 678 Ala Cys Phe Ile Gly Tyr Val Leu Pro Leu Ile Ile Ile Leu Ile Cys200 205 210 215 tat tct cag atc tgc tgc aaa ctc ttc aga act gcc aaa caaaac cca 726 Tyr Ser Gln Ile Cys Cys Lys Leu Phe Arg Thr Ala Lys Gln AsnPro 220 225 230 ctc act gag aaa tct ggt gta aac aaa aag gct ctc aac acaatt att 774 Leu Thr Glu Lys Ser Gly Val Asn Lys Lys Ala Leu Asn Thr IleIle 235 240 245 ctt att att gtt gtg ttt gtt ctc tgt ttc aca cct tac catgtt gca 822 Leu Ile Ile Val Val Phe Val Leu Cys Phe Thr Pro Tyr His ValAla 250 255 260 att att caa cat atg att aag aag ctt cgt ttc tct aat ttcctg gaa 870 Ile Ile Gln His Met Ile Lys Lys Leu Arg Phe Ser Asn Phe LeuGlu 265 270 275 tgt agc caa aga cat tcg ttc cag att tct ctg cac ttt acagta tgc 918 Cys Ser Gln Arg His Ser Phe Gln Ile Ser Leu His Phe Thr ValCys 280 285 290 295 ctg atg aac ttc aat tgc tgc atg gac cct ttt atc tacttc ttt gca 966 Leu Met Asn Phe Asn Cys Cys Met Asp Pro Phe Ile Tyr PhePhe Ala 300 305 310 tgt aaa ggg tat aag aga aag gtt atg agg atg ctg aaacgg caa gtc 1014 Cys Lys Gly Tyr Lys Arg Lys Val Met Arg Met Leu Lys ArgGln Val 315 320 325 agt gta tcg att tct agt gct gtg aag tca gcc cct gaagaa aat tca 1062 Ser Val Ser Ile Ser Ser Ala Val Lys Ser Ala Pro Glu GluAsn Ser 330 335 340 cgt gaa atg aca gaa acg cag atg atg ata cat tcc aagtct tca aat 1110 Arg Glu Met Thr Glu Thr Gln Met Met Ile His Ser Lys SerSer Asn 345 350 355 gga aag tgaaatggat tgtattttgg tttatagtga cgtaaactgtatgacaaact 1166 Gly Lys 360 ttgcaggact tcccttataa agcaaaataa ttgttcagcttccaattagt attcttttat 1226 atttctttca ttgggcgctt tcccatctcc aactcggaagtaagcccaag agaacaacat 1286 aaagcaaaca acataaagca caataaaaat gcaaataaatattttcattt ttatttgtaa 1346 acgaatacac caaaaggagg cgctcttaat aactcccaatgtaaaaagtt ttgttttaat 1406 aaaaaattaa ttattattct tgccaacaaa tggctagaaaggactgaata gattatatat 1466 tgccagatgt taatactgta acatactttt taaataacatatttcttaaa tccaaatttc 1526 tctcaatgtt agatttaatt ccctcaataa caccaatgttttgttttgtt tcgttctggg 1586 tcataaaact ttgttaagga actcttttgg aataaagagcaggatgctgc ggaattc 1643 4 361 PRT Homo sapiens 4 Met Asp Ile Gln Met AlaAsn Asn Phe Thr Pro Pro Ser Ala Thr Pro 1 5 10 15 Gln Gly Asn Asp CysAsp Leu Tyr Ala His His Ser Thr Ala Arg Ile 20 25 30 Val Met Pro Leu HisTyr Ser Leu Val Phe Ile Ile Gly Leu Val Gly 35 40 45 Asn Leu Leu Ala LeuVal Val Ile Val Gln Asn Arg Lys Lys Ile Asn 50 55 60 Ser Thr Thr Leu TyrSer Thr Asn Leu Val Ile Ser Asp Ile Leu Phe 65 70 75 80 Thr Thr Ala LeuPro Thr Arg Ile Ala Tyr Tyr Ala Met Gly Phe Asp 85 90 95 Trp Arg Ile GlyAsp Ala Leu Cys Arg Ile Thr Ala Leu Val Phe Tyr 100 105 110 Ile Asn ThrTyr Ala Gly Val Asn Phe Met Thr Cys Leu Ser Ile Asp 115 120 125 Arg PheIle Ala Val Val His Pro Leu Arg Tyr Asn Lys Ile Lys Arg 130 135 140 IleGlu His Ala Lys Gly Val Cys Ile Phe Val Trp Ile Leu Val Phe 145 150 155160 Ala Gln Thr Leu Pro Leu Leu Ile Asn Pro Met Ser Lys Gln Glu Ala 165170 175 Glu Arg Ile Thr Cys Met Glu Tyr Pro Asn Phe Glu Glu Thr Lys Ser180 185 190 Leu Pro Trp Ile Leu Leu Gly Ala Cys Phe Ile Gly Tyr Val LeuPro 195 200 205 Leu Ile Ile Ile Leu Ile Cys Tyr Ser Gln Ile Cys Cys LysLeu Phe 210 215 220 Arg Thr Ala Lys Gln Asn Pro Leu Thr Glu Lys Ser GlyVal Asn Lys 225 230 235 240 Lys Ala Leu Asn Thr Ile Ile Leu Ile Ile ValVal Phe Val Leu Cys 245 250 255 Phe Thr Pro Tyr His Val Ala Ile Ile GlnHis Met Ile Lys Lys Leu 260 265 270 Arg Phe Ser Asn Phe Leu Glu Cys SerGln Arg His Ser Phe Gln Ile 275 280 285 Ser Leu His Phe Thr Val Cys LeuMet Asn Phe Asn Cys Cys Met Asp 290 295 300 Pro Phe Ile Tyr Phe Phe AlaCys Lys Gly Tyr Lys Arg Lys Val Met 305 310 315 320 Arg Met Leu Lys ArgGln Val Ser Val Ser Ile Ser Ser Ala Val Lys 325 330 335 Ser Ala Pro GluGlu Asn Ser Arg Glu Met Thr Glu Thr Gln Met Met 340 345 350 Ile His SerLys Ser Ser Asn Gly Lys 355 360 5 1161 DNA Homo sapiens CDS 14..703 5gaattccgca gcc atg acc ccg cag ctt ctc ctg gcc ctt gtc ctc tgg 49 MetThr Pro Gln Leu Leu Leu Ala Leu Val Leu Trp 1 5 10 gcc agc tgc ccg ccctgc agt gga agg aaa ggg ccc cca gca gct ctg 97 Ala Ser Cys Pro Pro CysSer Gly Arg Lys Gly Pro Pro Ala Ala Leu 15 20 25 aca ctg ccc cgg gtg caatgc cga gcc tct cgg tac ccg atc gcc gtg 145 Thr Leu Pro Arg Val Gln CysArg Ala Ser Arg Tyr Pro Ile Ala Val 30 35 40 gat tgc tcc tgg acc ctg ccgcct gct cca aac tcc acc agc ccc gtg 193 Asp Cys Ser Trp Thr Leu Pro ProAla Pro Asn Ser Thr Ser Pro Val 45 50 55 60 tcc ttc att gcc acg tac aggctc ggc atg gct gcc cgg ggc cac agc 241 Ser Phe Ile Ala Thr Tyr Arg LeuGly Met Ala Ala Arg Gly His Ser 65 70 75 tgg ccc tgc ctg cag cag acg ccaacg tcc acc agc tgc acc atc acg 289 Trp Pro Cys Leu Gln Gln Thr Pro ThrSer Thr Ser Cys Thr Ile Thr 80 85 90 gat gtc cag ctg ttc tcc atg gct ccctac gtg ctc aat gtc acc gcc 337 Asp Val Gln Leu Phe Ser Met Ala Pro TyrVal Leu Asn Val Thr Ala 95 100 105 gtc cac ccc tgg ggc tcc agc agc agcttc gtg cct ttc ata aca gag 385 Val His Pro Trp Gly Ser Ser Ser Ser PheVal Pro Phe Ile Thr Glu 110 115 120 cac atc atc aag ccc gac cct cca gaaggc gtg cgc cta agc ccc ctc 433 His Ile Ile Lys Pro Asp Pro Pro Glu GlyVal Arg Leu Ser Pro Leu 125 130 135 140 gct gag cgc cag cta cag gtg cagtgg gag cct ccc ggg tcc tgg ccc 481 Ala Glu Arg Gln Leu Gln Val Gln TrpGlu Pro Pro Gly Ser Trp Pro 145 150 155 ttc cca gag atc ttc tca ctg aagtac tgg atc cgt tac aag cgt cag 529 Phe Pro Glu Ile Phe Ser Leu Lys TyrTrp Ile Arg Tyr Lys Arg Gln 160 165 170 gga gct gcg cgc ttc cac cgg gtgggg ccc att gaa gcc acg tcc ttc 577 Gly Ala Ala Arg Phe His Arg Val GlyPro Ile Glu Ala Thr Ser Phe 175 180 185 atc ctc agg gct gtg cgg ccc cgagcc agg tac tac gtc caa gtg gcg 625 Ile Leu Arg Ala Val Arg Pro Arg AlaArg Tyr Tyr Val Gln Val Ala 190 195 200 gct cag gac ctc aca gac tac ggggaa ctg agt gac tgg agt ctc ccc 673 Ala Gln Asp Leu Thr Asp Tyr Gly GluLeu Ser Asp Trp Ser Leu Pro 205 210 215 220 gcc act gcc aca atg agc ctgggc aag tagcaagggc ttcccgctgc 720 Ala Thr Ala Thr Met Ser Leu Gly Lys225 ctccagacag cacctgggtc ctcgccaccc taagccccgg gacacctgtt ggagggcgga780 tgggatctgc ctagcctggg ctggagtcct tgctttgctg ctgctgagct gccgggcaac840 ctcagatgac cgacttttcc ctttgagcct cagtttctct agctgagaaa tggagatgta900 ctactctctc ctttaccttt acctttacca cagtgcaggg ctgactgaac tgtcactgtg960 agatattttt tattgtttaa ttagaaaaga attgttgttg ggctgggcgc agtggatcgc1020 acctgtaatc ccagtcactg ggaagccgac gtgggtgggt agcttgaggc caggagctcg1080 aaaccagtcc gggccacaca gcaagacccc atctctaaaa aattaatata aatataaaat1140 aaaaaaaaaa aaaaggaatt c 1161 6 229 PRT Homo sapiens 6 Met Thr ProGln Leu Leu Leu Ala Leu Val Leu Trp Ala Ser Cys Pro 1 5 10 15 Pro CysSer Gly Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg 20 25 30 Val GlnCys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp 35 40 45 Thr LeuPro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala 50 55 60 Thr TyrArg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys Leu 65 70 75 80 GlnGln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu 85 90 95 PheSer Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His Pro Trp 100 105 110Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys 115 120125 Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg Gln 130135 140 Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu Ile145 150 155 160 Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly AlaAla Arg 165 170 175 Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe IleLeu Arg Ala 180 185 190 Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val AlaAla Gln Asp Leu 195 200 205 Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser LeuPro Ala Thr Ala Thr 210 215 220 Met Ser Leu Gly Lys 225 7 9 PRT Homosapiens UNSURE (2)..(2) Xaa = Ile or Val 7 Ser Xaa Asp Arg Xaa Xaa XaaXaa Xaa 1 5

What is claimed is:
 1. A DNA segment coding for a polypeptide having anamino acid sequence corresponding to a polypeptide selected from thegroup consisting of EBI 1, EBI 2, and EBI 3 polypeptides.
 2. The DNAsegment according to claim 1, wherein the DNA segment has a sequenceselected from the group consisting of sequences set forth in SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5; or allelic, mutant or speciesvariation thereof.
 3. The DNA segment according to claim 1, wherein theDNA segment has an allelic variation of a sequence selected from thegroup consisting of sequences set forth in SEQ ID NO:1, SEQ ID NO:3, andSEQ ID NO:5.
 4. The DNA segment according to claim 1, wherein the DNAsegment encodes an amino acid sequence selected from the groupconsisting of sequences set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQID NO:6, or mutant or species variations thereof.
 5. The DNA segmentaccording to claim 1, wherein the DNA segment has a sequence selectedfrom the group consisting of sequences set forth in SEQ ID NO:1, SEQ IDNO:3, and SEQ ID NO:5.
 6. The DNA segment according to claim 1, whereinthe DNA segment encodes an amino acid sequence selected from the groupconsisting of sequences set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQID NO:6.
 7. A substantially pure polypeptide having an amino acidsequence corresponding to a polypeptide selected from the groupconsisting of EBI 1, EBI 2, and EBI 3 polypeptides.
 8. The polypeptideaccording to claim 7, wherein the polypeptide has an amino acid sequenceselected from the group consisting of sequences set forth in SEQ IDNO:2, SEQ ID NO:4, and SEQ ID NO:6, or mutant or species variationthereof.
 9. A nucleic acid probe for the detection of the presence ofEpstein Barr Virus in a sample comprising the DNA segment according toclaim 1 or at least 20 contiguous nucleotides thereof.
 10. The nucleicacid probe according to claim 9, wherein the DNA segment has a nucleicacid sequence selected from the group consisting of sequences set forthin SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, or at least 20 contiguousnucleotides thereof.
 11. The nucleic acid probe according to claim 9,wherein the probe encodes an amino acid sequence selected from the groupconsisting of sequences set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQID NO:6, or at least 7 contiguous amino acids thereof.
 12. A method ofdetecting Epstein Barr Virus in a sample comprising: a) contacting saidsample with the nucleic acid probe according to claim 9, underconditions such that hybridization occurs, and b) detecting the presenceof said probe bound to RNA.
 13. A kit detecting the presence of EpsteinBarr virus in a sample comprising at least one container means havingdisposed therein the nucleic acid probe according to claim
 9. 14. Arecombinant DNA molecule comprising, 5′ to 3′, a promoter effective toinitiate transcription in a host cell and the DNA segment according toclaim
 1. 15. A cell that contains the DNA molecule according to claim14.
 16. A recombinant DNA molecule comprising a vector and the DNAsegment according to claim
 1. 17. A cell that contains the recombinantDNA molecule according to claim
 16. 18. A recombinant DNA moleculecomprising a transcriptional region functional in a cell, a sequencecomplimentary to an RNA sequence encoding an amino acid sequencecorresponding to the polypeptide of claim 7, and a transcriptionaltermination region functional in said cell.
 19. A cell that contains therecombinant DNA molecule according to claim
 18. 20. An antibody havingbinding affinity to a polypeptide having an amino acid sequence selectedfrom the group consisting of EBI 1, EBI 2, and EBI 3 polypeptide, or abinding fragment thereof.
 21. The antibody according to claim 20,wherein said polypeptide has an amino acid sequence selected from thegroup of sequences set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQ IDNO:6, or mutant or species variation thereof.
 22. The antibody accordingto claim 20, wherein said antibody is a monoclinal antibody.
 23. Amethod of detecting a polypeptide selected from the group consisting ofEBI 1, EBI 2, EBI 3 in a sample, comprising: a) contacting said samplewith an antibody according to claim 20, under conditions such thatimmunocomplexes form, and b) detecting the presence of said antibodybound to said polypeptide.
 24. A diagnostic kit comprising: I) a firstcontainer means containing the antibody according to claim 20, and ii)second container means containing a conjugate comprising a bindingpartner of said monoclinal antibody and a label.
 25. A hybridoma whichproduces the monoclonal antibody according to claim 22, or bindingfragment thereof.
 26. The hybridoma according to claim 25, wherein saidpolypeptide has an amino acid sequence selected from the group ofsequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or mutantor species variation thereof, or at least 7 contiguous amino acidsthereof.